Development of operational immunologic tolerance with neonatal gene transfer in nonhuman primates: preliminary studies.

Achieving persistent expression is a prerequisite for effective genetic therapies for inherited disorders. These proof-of-concept studies focused on adeno-associated virus (AAV) administration to newborn monkeys. Serotype rh10 AAV expressing ovalbumin and green fluorescent protein (GFP) was administered intravenously at birth and compared with vehicle controls. At 4 months postnatal age, a second injection was administered intramuscularly, followed by vaccination at 1 year of age with ovalbumin and GFP. Ovalbumin was highest 2 weeks post administration in the treated monkey, which declined but remained detectable thereafter; controls demonstrated no expression. Long-term AAV genome copies were present in myocytes. At 4 weeks, neutralizing antibodies to rh10 were present in the experimental animal only. With AAV9 administration at 4 months, controls showed transient ovalbumin expression that disappeared with the development of strong anti-ovalbumin and anti-GFP antibodies. In contrast, increased and maintained ovalbumin expression was noted in the monkey administered AAV at birth, without antibody development. After vaccination, the experimental monkey maintained levels of ovalbumin without antibodies, whereas controls demonstrated high levels of antibodies. These preliminary studies suggest that newborn AAV administration expressing secreted and intracellular xenogenic proteins may result in persistent expression in muscle, and subsequent vector administration can result in augmented expression without humoral immune responses.

Overexpression of transforming growth factor-beta1 in fetal monkey lung results in prenatal pulmonary fibrosis.

Altered transforming growth factor (TGF)-ß expression levels have been linked to a variety of human respiratory diseases, including bronchopulmonary dysplasia and pulmonary fibrosis. However, a causative role for aberrant TGF-ß in neonatal lung diseases has not been defined in primates. Exogenous and transient TGF-ß1 overexpression in fetal monkey lung was achieved by transabdominal ultrasound-guided fetal intrapulmonary injection of adenoviral vector expressing TGF-ß1 at the second or third trimester of pregnancy. The lungs were then harvested near term, and fixed for histology and immunohistochemistry. Lung hypoplasia was observed where TGF-ß1 was overexpressed during the second trimester. The most clearly marked phenotype consisted of severe pulmonary and pleural fibrosis, which was independent of the gestational time point when TGF-ß1 was overexpressed. Increased cell proliferation, particularly in α-smooth muscle actin-positive myofibroblasts, was detected within the fibrotic foci. But epithelium to mesenchyme transdifferentiation was not detected. Massive collagen fibres were deposited on the inner and outer sides of the pleural membrane, with an intact elastin layer in the middle. This induced fibrotic pathology persisted even after adenoviral-mediated TGF-ß1 overexpression was no longer evident. Therefore, overexpression of TGF-ß1 within developing fetal monkey lung results in severe and progressive fibrosis in lung parenchyma and pleural membrane, in addition to pulmonary hypoplasia.

Tissue-specific restriction of cyclophilin A-independent HIV-1- and SIV-derived lentiviral vectors.

The host factor alpha isoform of the tripartite motif 5 (TRIM5alpha) restricts human immunodeficiency virus type 1 (HIV-1) infection in certain non-human primate species. Restriction of HIV-1 is enhanced by binding of the viral capsid to cyclophilin A (CypA) in target cells, although CypA is not absolutely required for restriction in rhesus macaque cells. Simian immunodeficiency virus (SIV) is not restricted by rhesus macaque TRIM5alpha and its capsid does not bind to CypA. Here, the effect of lentiviral CypA dependence on restriction in different tissues was examined by engineering an HIV-1 capsid quadruple mutant (V(86)P/H(87)Q/I(91)V/M(96)I) lentiviral vector (HIV(quad)) that is CypA-independent. Whereas HIV-1 was restricted in rhesus macaque and owl monkey epithelial cells, infection with the HIV(quad) vector was efficient at high viral concentrations. In contrast, HIV(quad) was largely restricted in primary rhesus macaque CD34(+) cells. Human epithelial and primary CD34(+) cells were permissive for HIV-1, HIV(quad) and SIV, whereas transduction of human T cells by HIV(quad) or SIV was impaired. The restrictive human cells did not express increased levels of TRIM5alpha, and restriction was not relieved by abolishing CypA, consistent with HIV(quad) and SIV being CypA-independent. Pseudotyping of lentiviral vectors with the gibbon ape leukemia virus envelope altered their sensitivity to perturbations of the virus-CypA interaction compared to pseudotyping with vesicular stomatitis virus glycoproteins, suggesting that the viral entry pathway modulates restriction. Together, these studies reveal that an HIV-1 capsid quadruple mutant can partially overcome lentiviral restriction in non-human primate epithelial cells, but not in hematopoietic cells. Similarly, human cells vary in their permissiveness for CypA-independent lentiviruses, and suggest the presence of tissue-specific factor(s) that can inhibit lentiviral transduction independently of viral interaction with TRIM5alpha and CypA.

Direct administration of insulin-like growth factor to fetal rhesus monkeys (Macaca mulatta).

A potential treatment for the amelioration of fetal growth failure is insulin-like growth factor-I (IGF-I). To address concerns of safety and efficacy, IGF-I (80 microg/kg; GroPep Pty.) was administered i.p. to healthy rhesus monkey fetuses via ultrasound guidance every other day between gestational days (GD) 110-120 and 130-140 (third trimester; term = approximately GD 165 +/- 10; n = 6). Pregnancies were monitored sonographically, and fetal/maternal blood samples were collected for complete blood counts, immunophenotyping, and biochemical analyses. Blood samples, external measures of the fetus and newborn, and tissue and organ weights were collected at fetal necropsy (GD 150; n = 2) or at term delivery of neonates (GD 160; n = 4). The results of these investigations have shown no evidence of hypoglycemia in the fetus or dam during the course of treatment. Circulating concentrations of fetal, but not maternal, IGF-I increased with treatment (approximately 80 to approximately 1015 ng/ml), and there was no evidence of a change in serum IGF-II or an increase in IGF binding protein-3 compared with historical control values. Fetal lymphocytes and select red cell parameters increased, and a significant elevation in circulating B cells and CD4/CD8 ratios in fetal lymph nodes was shown. Although no changes were detected in body weights, increases in thymic, splenic, and kidney weights and small intestine lengths occurred. Thus, administration of IGF-I to the fetal monkey is safe and results in 1) transient increases in circulating IGF-I, 2) a significant effect on fetal hematopoietic and lymphoid tissues, and 3) an increase in select fetal organ weights and measures. These data suggest that IGF-I may represent a potential candidate for therapeutic treatment of growth-compromised human fetuses in utero.

A role for epidermal growth factor in the morphological and functional maturation of the adrenal gland in the fetal rhesus monkey in vivo.

We determined the effects of epidermal growth factor (EGF) and beta-methasone on the growth and development of the adrenal gland of the fetal rhesus monkey in vivo between 121-128 days of gestation. The adrenal to body weight ratio was significantly greater (P < 0.05) in EGF-treated fetuses (0.988 +/- 0.046 x 10(-3) g/g) and significantly reduced (P < 0.05) in beta-methasone-treated fetuses (0.401 +/- 0.056 x 10(-3) g/g) compared with that in control fetuses (0.689 +/- 0.050 x 10(-3) g/g). The increase in adrenal weight with EGF administration was due to hypertrophy of definitive zone cells of the adrenal cortex, whereas the reduction in adrenal weight after beta-methasone treatment was due to a decrease in the size of definitive and fetal zone cells of the adrenal cortex. By Western analysis, EGF treatment induced a significant (P < 0.05) 2.8-fold increase in the amount of protein for 3 beta-hydroxysteroid dehydrogenase/isomerase (3 beta HSD) in the fetal adrenal. EGF also stimulated the induction of immunocytochemical staining for 3 beta HSD in transitional zone cells of the adrenal cortex. In contrast, beta-methasone resulted in 2.6-, 4.5-, and 6.6-fold significant decreases (P < 0.05) in the amount of protein for cytochrome P450 cholesterol side-chain cleavage, cytochrome P450 17 alpha-hydroxylase/17,20-lyase, and 3 beta HSD in the fetal adrenal. After beta-methasone treatment. 3 beta HSD staining was detected in some of the definitive zone cells, with no 3 beta HSD staining in the transitional zone. In conclusion, growth and functional differentiation of fetal primate adrenal gland can be accelerated prematurely by EGF and inhibited by glucocorticoid negative feedback.

Ultrasound evaluation of fetuses of zinc-deprived monkeys (Macaca mulatta).

Fetal body movements were studied in three groups of gravid rhesus macaques fed different amounts of dietary zinc (100 micrograms Zn/g diet, control, n = 12; 4 micrograms Zn/g diet, marginal deprivation, n = 7; 2 micrograms Zn/g diet, moderate deprivation, n = 11). Sonographic examinations were conducted during the third trimester in awake chair-restrained dams. Movement categories, derived from the human biophysical profile, were motor activity (trunk and limb movements), startle, and breathing movements. Moderately deprived fetuses were more active than controls on gestational day (GD) 115; maternal plasma zinc concentrations were significantly correlated with fetal activity at this time. In addition moderately deprived fetuses exhibited fewer breathing episodes on GDs 115-135. Biometrics measures indicated growth retardation in one moderately deprived fetus. These data suggest that moderate, but not marginal, dietary zinc deprivation influences fetal status as evaluated by sonography.

Transplantation of human peripheral blood stem cells into fetal rhesus monkeys (Macaca mulatta).

BACKGROUND: Methods for assessing engraftment efficiency have been explored in a primate xenogeneic model of in utero hematopoietic stem cell transplantation. METHODS: Human peripheral blood stem cells (PBSC) were obtained by leukapheresis from a human male donor after 4 days of administration of recombinant human granulocyte-colony stimulating factor (5 microg/kg/ day). PBSC were enriched for the CD34+ population with and without T-cell depletion. The resulting mononuclear cells consisted of two cell populations, one that was stem cell enriched (0.83% CD3+ cells, 95% CD34+; group 1) and one that was stem cell enriched and T-cell depleted (<0.03% CD3+ cells, 98% CD34+; group 2). Four fetal monkeys (two per group) received either two or four i.p. injections (approximately 5x10(6) cells/injection) via ultrasound guidance every other day over a 7-day period (gestational days 50, 52, 54, and 56). One fetus in each group also received i.p. recombinant human stem cell factor (25 microg/kg) and recombinant human granulocyte-colony stimulating factor (10 microg/kg) posttransplant every 10 days from gestational day 60-150. RESULTS: Four healthy newborns were delivered at term, and specimens were analyzed by polymerase chain reaction for the human Y chromosome (birth, monthly to 6 months; blood, marrow, progenitor assays). Polymerase chain reaction results were positive for all four newborns in all specimens assessed, and flow cytometric analysis for human CD45 in marrow showed engraftment ranging from 0.1-1.7%. There was no evidence of graft-versus-host disease in any of the animals. CONCLUSION: These studies show that (1) multilineage engraftment of human PBSC can be achieved in the fetal rhesus recipient, (2) the rhesus fetus appears to tolerate relatively high numbers of human CD3+ cells, and (3) healthy chimeric rhesus infants can be delivered at term after multiple in utero procedures.

Real-time TaqMan PCR as a specific and more sensitive alternative to the branched-chain DNA assay for quantitation of simian immunodeficiency virus RNA.

We developed a rapid and highly reproducible assay based on real-time PCR (TaqMan, Applied Biosystems, Foster City, CA) to quantitate simian immunodeficiency virus (SIV) RNA in plasma samples. This assay was compared with the current branched-chain DNA assay (Bayer, Emeryville, CA). Results obtained with the real-time TaqMan PCR assay were comparable to those obtained with the branched-chain DNA assay in overlapping ranges of sensitivities (r = 0.9429, p < 0.05). However, the real-time TaqMan PCR assay was capable of detecting as few as 50 copies of RNA/ml, whereas branched-chain DNA was only sensitive to 1,500 copies of RNA/ml. Therefore, several animals that tested negative by branched-chain DNA were positive by realtime TaqMan PCR. Two false positive tests were also recorded for the branched-chain DNA test. False negative and positive tests were confirmed by cell culture isolation and conventional nested RT-PCR. The SIV TaqMan assay detected a wide range of wild-type, cloned, and recombinant SIV strains with similar amplification efficiency, including SIVmac251, SIVmac239, SIVmac239 containing the 184V mutation in RT, SIV1A11, SIVmac239 delta3, SIVmac-M4, and chimeras (SHIVs) containing specific HIV-1 genes, such as reverse transcriptase (RT-SHIV) or Env (SHIV-E). In conclusion, the high sensitivity, increased specificity, wide dynamic range, simplicity, and reproducibility of the real-time SIV RNA quantitation allow the screening of large numbers of samples and make this method especially suitable for measuring both viral DNA and RNA levels during vaccine and therapy studies.

Intervillous blood flow in the third trimester gravid rhesus monkey (Macaca mulatta): use of sonographic contrast agent and harmonic imaging.

A properly implanted and functioning placenta is essential for the normal outcome of pregnancy. As pregnancy advances, an increasing supply of maternal blood, which reaches the intervillous space of the placenta via the spiral arteries, is necessary for continued growth and development of the fetus. Presumably, deficient blood flow to the intervillous space can lead to placental ischaemia and an unfavourable outcome, such as pre-eclampsia. In this study, we used a primate model, where echocontrast-enhanced harmonic imaging was utilized to demonstrate placental intervillous blood flow without visualization of fetal blood circulation within the chorionic villi. We propose that this technique, which requires further assessment of efficacy and safety prior to use in humans, is a potentially useful non-invasive clinical tool for assessing intervillous blood flow in the third trimester of pregnancy.

Long-term engraftment following in utero T cell-depleted parental marrow transplantation into fetal rhesus monkeys.

A major concern with allogeneic BMT for treating most inherited diseases is the need to overcome graft rejection with conditioning chemotherapy which is associated with a relatively high morbidity and mortality. This can be eliminated if the transplant is done in utero when the fetus is unable to reject donor hematopoietic stem cells (HSC). We studied the efficacy of T cell-depleted (TCD) parental bone marrow as a source of HSC for transplantation into early gestation non-defective fetal rhesus monkeys. Thirteen opposite sexed TCD transplants were done into 44 day fetal recipients and 12 into 61 day recipients (165 day total gestation). The procedure-related mortality was 8%, all in the earlier age group. The overall survival was 60% at birth with a projected survival of 44 +/- 10% at 1.5 years with no difference between the two age groups. We used a PCR assay for the rhesus Y chromosome to detect male donor cells in female recipients (six animals transplanted at 44 days and five at 63 days). The overall engraftment rate was 73% with no difference as a function of gestational age at transplant. In six long-term surviving engrafted females we detected donor cells in the peripheral blood and bone marrow up to 3 years of age. We found a delay in the appearance of donor cells in the peripheral blood in engrafted animals, in some cases for up to 6 months post-BMT. In vitro mixed lymphocyte reaction and cell-mediated lymphocytotoxicity studies between the recipient and donor cells indicate that tolerance was induced to donor cells. Individual and pooled erythroid and myeloid marrow colonies grown in methyl cellulose were collected and analyzed for donor origin by PCR. The amount of donor cells in marrows from long-term engrafted animals was < 0.1%. In a fetal recipient studied at 35 days post-BMT, donor cells were detected in bone marrow and liver in both erythroid and myeloid lineages. These results indicate that TCD parental marrow can durably engraft in utero. While the engraftment rate is similar to that seen with fetal liver as the source of HSC, the degree of peripheral blood engraftment (percent donor cells) in this non-defective primate model is low. It will require increasing the percent pre-or postnatally for this approach to be clinically relevant in those disorders in which there is no selective survival advantage for normal engrafted donor cells.

The missing link in rhesus monkey amniotic fluid volume regulation: intramembranous absorption.

OBJECTIVE: To investigate whether intramembranous absorption occurs in the rhesus monkey and its role in amniotic fluid (AF) volume regulation as a possible model for the human fetus. MATERIALS AND METHODS: We studied five chronically catheterized rhesus monkey fetuses (Macaca mulatta) at 126 +/- 1 (standard error) days' gestation (term approximately 165 days) with ligated esophagi and catheterized tracheae. Samples (0.5 mL each) of fetal and maternal blood and amniotic and lung fluid were collected at 0, 15, 30, 60, 120, 180, and 240 minutes after injection of 0.1 mCi (3 mL) of technetium-99m (Tc-99m) into the amniotic cavity. RESULTS: In spite of esophageal ligation, there was a rapid absorption of the Tc-99m into the fetal circulation within 15 minutes of injection. The maternal Tc-99m activity increased in parallel to fetal activity but remained lower. The fetal lung fluid Tc-99m activity increased more slowly but was equivalent to the fetal circulating level by 4 hours. CONCLUSIONS: These results suggest that intramembranous absorption occurs and may play an important role in rhesus AF volume regulation and composition. Furthermore, this animal model, which closely resembles the human, may provide valuable insight into abnormalities of human AF volume regulation.

Fetal monkey surfactants after intra-amniotic or maternal administration of betamethasone and thyroid hormone.

OBJECTIVE: To compare direct intra-amniotic injection of betamethasone and thyroxine (T4) with maternal treatment and controls for accelerating pulmonary surfactant production. METHODS: Twelve pregnant monkeys (Macaca mulatta) on gestational day 125 (term 165 +/- 10 days) had surfactant protein A and B concentrations measured in amniotic fluid. In four controls, normal saline was injected into the amniotic fluid; four others (intra-amniotic) received intra-amniotic betamethasone (1 mg) and T4 (60 microg); and in four others (maternal), the dam was given betamethasone (12 mg) intramuscularly, repeated in 24 hours, plus TRH (400 microg) intravenously, repeated every 6 hours for 24 hours. Seventy-two hours after the initial amniocentesis, a hysterotomy was performed and fetal tissue and amniotic fluid harvested for determination of surfactant protein A and B concentrations and immunohistochemical staining for surfactant protein A. RESULTS: Amniotic fluid surfactant protein A was higher with intra-amniotic injection than with maternal treatment (P <.04) or controls (P =.07). Amniotic fluid surfactant protein B was higher in the intra-amniotic group than in controls (P =.06). Immunohistochemical staining for surfactant protein A in the lung tissue was increased in the intra-amniotic group compared with controls (0.145 +/- 0.01 versus 0.097 +/- 0.001, percent positive staining for surfactant protein A cells per lung tissue cells; P <.03). Birth weight was greater in the intra-amniotic group compared with the maternal group (P <.03) although not different from the controls. Finally, gut motility and the presence of formed meconium were increased in the intra-amniotic group compared with the other groups (P <.05). CONCLUSION: Intra-amniotic injection of betamethasone and T4 enhanced lung (and possibly intestinal) maturation of the preterm rhesus fetal monkey compared with maternal injections.

Plasmodium coatneyi in the rhesus monkey (Macaca mulatta) as a model of malaria in pregnancy.

Pregnant women with Plasmodium falciparum infection are at increased risk for complications such as anemia and cerebral malaria. In addition, the infants of these women suffer intrauterine growth retardation (IUGR), low birth weight (LBW), congenital infection, and high infant mortality. Although much has been learned from studies of malaria during human pregnancy, progress has been limited by the lack of a suitable animal model. Nonhuman primates are of particular interest because, other than the armadillo, they are the only animals with a discoidal, villous, hemochorial placenta like that of humans. We have established a model of malaria during human pregnancy by inoculating pregnant rhesus monkeys (Macaca mulatta) with Plasmodium coatneyi (a sequestering parasite) during the first trimester. In our initial experiment, four monkeys were inoculated with a fresh inoculum containing 10(8) viable parasites from an infected donor monkey. All four monkeys became parasitemic seven days postinoculation (PI) and three monkeys aborted 7-10 days PI coincident with high peak parasitemias (41,088-374,325 parasites/mm3). Although abortion is one of the outcomes observed in Plasmodium-infected women, the intent of this study was to examine the effects of Plasmodium infection throughout gestation. Since the rapid onset of high parasitemia may have been responsible for the abortions, a decision was made to reduce the size of the effective inoculum. Six additional pregnant monkeys were inoculated with a frozen isolate taken from the same donor containing 10(6) parasites. These six animals became parasitemic by 14 days PI and, along with monkey E412, carried their infants to term. These seven infants weighed significantly less at term than the infants of uninfected mothers (P = 0.0355). Symmetrical IUGR was detected by ultrasound in one fetus with an LBW of 334 g. Another LBW infant (300 g) had asymmetrical growth retardation, which has been associated with uteroplacental insufficiency and was consistent with the lower placental weights found in infected dams compared with controls (P = 0.0455). The infant with symmetric IUGR died at five days of age, while the other is alive but congenitally infected. The IUGR, LBW, congenital infection, postnatal infant mortality, and early abortions observed in these animals suggest that P. coatneyi in pregnant rhesus monkeys is a valid model of malaria in human pregnancy. This model should provide the opportunity to study questions about malaria in pregnancy that have been difficult to study in humans.

Incorporation of a micronucleus study into a developmental toxicology and pharmacokinetic study of L-selenomethionine in nonhuman primates.

Concomitant to a developmental toxicology study of selenium in long-tailed macaques (Macaca fascicularis), a transplacental bone marrow micronucleus assay was conducted in the fetuses of treated animals. Selenium was administered as L-selenomethionine by nasogastric intubation at 0, 150 or 300 micrograms/kg-day to pregnant macaques daily throughout organogenesis (gestation days 20-50). Pregnancy was terminated on gestation day 100 +/- 2 and fetuses were obtained by hysterotomy. Selenium concentrations in maternal blood were monitored throughout pregnancy and selenium concentrations in fetal blood were measured at hysterotomy. Maternal circulating selenium did not exceed 4 ppm in plasma or 3.7 ppm in erythrocytes. Selenium in cord blood was < or = 0.1 ppm in plasma and < or = 1.1 ppm in erythrocytes at 300 micrograms/kg-day. Fetal bone marrow smears were prepared from the humerus and micronucleated polychromatic erythrocytes were scored. No increase of micronucleus frequency was detected in any dose group, although signs of maternal selenosis were obvious. This finding is compared to the previous observation that micronuclei were induced in the bone marrow of adult nonpregnant macaques treated at 600 micrograms/kg-day, a lethal dose yielding blood selenium levels to 7.3 ppm in plasma and 5.7 ppm in erythrocytes after 15 days of daily treatment, when death occurred. These data demonstrate that measurement of circulating xenobiotics can be useful for the interpretation of genetic toxicology results.

The effect of the anti-progestin RU 486 on early pregnancy in the long-tailed macaque (Macaca fascicularis).

The efficacy of various doses of RU 486 in terminating pregnancy before and after the luteal-placental shift (LPS) in the long-tailed macaque (Macaca fascicularis) was assessed through sonographic examination and measurements of steroid hormones and their metabolites. Intramuscular injection of 1.0, 2.5, 12.5, or 25.0 mg/kg was administered either from gestational day (GD) 15-18 (Group 1; N = 11) or GD 23-26 (Group 2; N = 9). The timing of treatment was determined by the detection of the preovulatory estrogen peak via daily urinary estrone conjugate (E1C) measurements. In Group 1, a 90.9% pregnancy loss was observed (10/11); seven animals aborted during GD 15-20, two animals indicated early embryonic death with retained gestational sacs, one animal aborted on GD 56, and one pregnancy was maintained. In Group 2, an 88.9% pregnancy loss was observed (8/9); eight animals aborted between GD 26-29, and one pregnancy was unaffected. Hormone profiles appeared to fall secondarily to the loss of trophoblast function. These results indicate: (a) RU 486 was more effective after the LPS; and (b) the primary effect of RU 486 appeared to be at the level of the products of conception.

Tamoxifen as an antifertility agent in the long-tailed macaque (Macaca fascicularis).

The potential use of tamoxifen, a nonsteroidal antiestrogen, as an antifertility agent was studied in the long-tailed macaque (Macaca fascicularis). Twenty-six cycling females were bred, then treated with a single oral dose of tamoxifen (5 mg/kg) (N = 13) or vehicle (N = 13) on day 4 post-ovulation. Serum progesterone (P4) and tamoxifen concentrations were evaluated on post-ovulation days 4, 8, 12, 16, and 18. No effects of treatment were observed on P4 concentrations or on the fertility rate--pregnancy was achieved in 4/13 controls (31%) and 6/13 treated females (46%). Analysis for serum tamoxifen concentrations in samples collected during the fertility and a supplemental pharmacokinetic study (N = 3; single oral dose of 10 mg/kg; urine and serum evaluated) failed to reveal any detectable tamoxifen levels. It was concluded that (1) absorption of tamoxifen may be negligible under the described treatment regimens or (2) tamoxifen metabolism/clearance occurs at a rapid rate.

Effects of two antiprogestins on early pregnancy in the long-tailed macaque (Macaca fascicularis).

The abortifacient effects of mifepristone and HRP 2000 were compared in gravid long-tailed macaques. Thirty-six animals were studied with treatment administered either by the oral (0.5 or 5.0 mg/kg; N = 5 per antiprogestin per dose) or intramuscular (i.m.) routes (0.5 mg/kg; N = 5 per antiprogestin) on gestational days (GD) 23-26; six vehicle controls were included. Blood samples were collected for assay of progesterone (P4) and each of the antiprogestins (pre-treatment, daily GD 23-28, every other day GD 30-40), and animals were monitored sonographically throughout gestation. Results of these studies indicated high rates of abortion with i.m. administration (3/5 mifepristone, 4/5 HRP 2000) and 5.0 mg/kg oral route (4/5, 2/5, respectively), with less effects noted at oral doses of 0.5 mg/kg (2/5, 0/5, respectively). No early abortions were observed in the control groups. Following daily i.m. treatment, peak levels of 8-16 ng/ml mifepristone were detected whereas 6-10 ng/ ml of HRP 2000 were noted (GD 26-27). No serum levels of mifepristone were detected following either of the oral doses whereas serum levels of 2-6 ng/ml HRP 2000 were noted with high dose oral administratation. Results of these studies suggest: (1) both antiprogestins are roughly comparable in terminating early pregnancy although HRP 2000 may be more efficacious when administered i.m. whereas mifepristone may be more effective when administered orally; (2) similar levels of biological activity are seen with the i.m. and high dose oral dosing regimens, with little or no activity with the oral low dose; and (3) infants resulting from surviving pregnancies were not affected by early gestation exposure.

PIP: The primary objective was to compare the relative potencies of mifepristone and another newly synthesized antiprogestin, HRP 2000 (17-alpha-acetoxy-11-beta-[4-N,N-dimethylaminophenyl]-pregna-4,9- diene-3,20-dione) in terminating early pregnancy in gravid long-tailed macaques. 36 animals were studied with treatment administered either by the oral (0.5 or 5.0 ng/kg; N = 5 per antiprogestin per dose) or intramuscular (im) routes (0.5 ng/kg; N = 5 per antiprogestin) on gestational days (GD) 23-26; 6 vehicle controls were included. Blood samples were collected for assay of progesterone (P4) and each of the antiprogestins (pre-treatment, daily GD 23-28, every other day GD 30-40), and the animals were monitored sonographically throughout gestation. Results of these studies indicated high rates of abortion with im administration (3/5 mifepristone, 4/5 HRP 2000) and the 5.0 mg/kg oral route (4/5, 2/5, respectively), with less effects noted at oral doses of 0.5 mg/kg (2/5, 0/5, respectively). No early abortions were observed in the control groups. Following daily im treatment, peak levels of 8-16 ng/ml mifepristone were detected, whereas 6-10 ng/ml of HRP 2000 were noted (GD 26-27). No serum levels of mifepristone were detected following either of the oral doses, whereas serum levels of 2-6 ng/ml HRP 2000 were noted with high-dose oral administration. Results of these studies suggest: 1) both antiprogestins are roughly comparable in terminating early pregnancy, although HRP 2000 may be more efficacious when administered im, whereas mifepristone may be more effective when administered orally; 2) similar levels of biological activity are seen with the im and high-dose oral dosing regimens, with little or no activity with the oral low dose; and 3) infants resulting from surviving pregnancies were not affected by early gestation exposure.

Ultrasound-induced lung hemorrhage in the monkey.

Studies with the mouse have shown that lung hemorrhage can result from exposure to ultrasound at a peak pressure of approximately 1 MPa at 4 MHz (Mechanical Index [MI] approximately 0.5). In order to determine whether a comparable outcome could occur in a larger animal with characteristics similar to humans, studies were performed with monkeys using a clinical scanner under maximum output conditions (imaging + pulsed and color Doppler; derated pr of 3.7 MPa [4.5 MPa, measured in water], 4 MHz; MI approximately 1.8) (N = 57). Monkeys ranged in age from 1 day of life to 16 years with exposures limited to the right lung lobes (5 min cranial, 5 min caudal; N = 41 exposed, N = 12 sham-exposed controls, N = 4 colony controls). Results showed that animals ranging in age from 3 months to 5 years (mean age of 2.5 years) had a greater propensity for the occurrence of multiple well-demarcated circular hemorrhagic foci (0.1-1.0 cm), which were not observed in either control group. These lesions were characterized by marked congestion of alveolar capillaries with accumulation of red blood cells within the alveolar spaces, and were unassociated with major vessels or respiratory bronchioles. Further studies will be required in order to determine the relevance of these findings to the human, although it was concluded that ultrasound-induced lung hemorrhage in the monkey is of a significantly lesser degree when compared to the mouse.

Hematologic and growth-related effects of frequent prenatal ultrasound exposure in the long-tailed macaque (Macaca fascicularis).

Prior investigations have shown that reduced birth weights and transient neutropenias result from frequent exposure of monkey fetuses to ultrasound. To further explore these findings, 26 animals were studied (16 exposed, 10 controls; "triple mode"; ATL Ultramark 9 with HDI; ISPTAd approximately 645 to 714 mW/cm2). Exposures were performed daily for 5 days each week from gestational days (GD) 21 to 35 (5 min), three times weekly from GD 36 to 60 (5 min), then weekly from GD 61 to 153 +/- 1 (10 min). Fetal blood samples (FBS) were collected for complete blood counts (CBCs), hematopoietic progenitor assay, circulating insulin-like growth factors (IGF-I, IGF-II) and binding proteins (IGFBP-3) (GD 120, 140, 153 +/- 1). Animals were delivered by Cesarean section at term (GD 153 +/- 1), and body weights, morphometrics, CBCs, and bone marrow aspirates assessed at delivery and postnatally for 3 months. Fetal neutropenias were noted in exposed animals in addition to reduced circulating progenitors (colony forming unit-granulocyte-macrophage [CFU-GM]). Growth of CFU-GM from bone marrow was exuberant at term, whereas circulating levels were diminished comparable to prenatal samples. Exposed animals were smaller at birth; marked reductions in IGFBP-3 were noted prenatally. These data suggest that frequent prenatal ultrasound exposure can transiently alter the neutrophil lineage, although these findings may be the result of enhanced margination and organ sequestration. Data also suggest that transient, altered growth patterns may be due to perturbations of the IGF axis.

Prenatal growth in the cynomolgus and rhesus macaque (Macaca fascicularis and Macaca mulatta): A comparison by ultrasonography.

The rhesus monkey has played an important role in the history of reproductive research. Because of limitations on the exportation and availability of this species, several other species of nonhuman primates have been considered as alternate models. Among them is the crab-eating, or cynomolgus, macaque (Macaca fascicularis), which displays similarities in developmental, reproductive, and physiological parameters. The use of both species of macaques for pregnancy-related studies necessitates the assessment of differences in growth and development through gestation. Observations during the embryonic and fetal periods in both species have been compared with diagnostic ultrasound. Results indicate no significant differences in size during the embryonic and early fetal periods, but a greater acceleration of growth in the rhesus begins at approximately 100-110 gestational days (GD). Analysis of embryonic and fetal heart rates indicate no differences between the two species. Normal predictive values for a variety of growth parameters including gestational sac, greatest length, biparietal diameter, and femur length have boon calculated by multiple regression analysis. These charts have proven useful for confirming the gestational age after timed matings and for predicting the age of animals for which the conception date is not known.