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.

Collecting duct epithelial-mesenchymal transition in fetal urinary tract obstruction.

Renal interstitial fibrosis contributes to the progression of most chronic kidney diseases and is an important pathologic feature of urinary tract obstruction. To study the origin of this fibrosis, we used a fetal non-human primate model of unilateral ureteric obstruction focusing on the role of medullary collecting duct (CD) changes. Obstruction at 70 days gestation (full term approximately 165 days) results in cystic dysplasia with significant medullary changes including loss of the epithelial phenotype and gain of a mesenchymal phenotype. These changes were associated with disruption of the epithelial basement membrane and concomitant migration of transitioning cells presumed responsible for the observed peritubular collars of fibrous tissue. There was an abundance of cells that co-expressed the intercalated cell marker carbonic anhydrase II and smooth muscle actin. These cells migrated through the basement membrane and were significantly reduced in obstructed ducts with peritubular collars. Our studies suggest that fetal urinary tract obstruction results in a CD epithelial-mesenchymal transition contributing to the interstitial changes associated with poor prognosis. This seems restricted to the intercalated cells, which contribute to the expansion of the principal cell population and the formation of peritubular collars, but are depleted in progressive injury.

Gene therapy to inhibit xenoantibody production using lentiviral vectors in non-human primates.

Xenoantibodies to the gal alpha1,3 gal (gal) epitope impede the use of pig tissues for xenotransplantation, a procedure that may help overcome the shortage of human organ donors. Stable gal chimerism and tolerance to gal(+) hearts could be achieved in alpha1,3-galactosyltransferase (alpha1,3GT)(-/-) mice using lentiviral vectors expressing porcine alpha1,3GT, the enzyme that synthesizes the gal carbohydrate. In this study, we evaluated whether chimerism sufficient to inhibit anti-gal xenoantibody responses can be achieved using lentivectors in non-human primates. Rhesus macaques were transplanted with autologous, alpha1,3GT-transduced bone marrow (BM) following sublethal irradation. Simian immunodeficiency virus (SIV)- and human immunodeficiency virus (HIV)-1-derived lentiviral constructs were compared. Chimerism was observed in several hematopoietic lineages in all monkeys. Engraftment in animals receiving SIV-based alpha1,3GT constructs was similar to that achieved using the HIV-1-derived lentivector for the first 2 months post-transplantation, but increased thereafter to reach higher levels by 5 months. Upon immunization with porcine hepatocytes, the production of anti-gal immunoglobulin M xenoantibody was substantially reduced in the gal(+) BM recipients compared to controls. This study is the first to report the application of gene therapy to achieve low-level, long-term gal chimerism sufficient to inhibit production of anti-gal antibodies after immunization with porcine cells in rhesus macaques.

HIV-1-derived lentiviral vectors and fetal route of administration on transgene biodistribution and expression in rhesus monkeys.

The gene transfer efficiency of lentiviral vectors pseudotyped with vesicular stomatitis virus-glycoprotein (VSV-G) driven by the MND or CMV promoters and expressing the enhanced green fluorescent protein (EGFP) was investigated in fetal rhesus monkeys (Macaca mulatta) (N=21). Fetuses (50+/-10 days gestation; term 165+/-10 days) were injected under ultrasound guidance using an intraperitoneal (i.p.) or intrahepatic (i.h.) approach with a range of 1 x 10(7)-2 x 10(8) infectious particles/fetus. Analysis of transgene biodistribution and expression was performed in multiple tissues at 3-7 months postgene delivery using quantitative techniques. Overall, results indicated the following: (1) i.p. gene transfer at 40 days gestation resulted in a more diffuse distribution of the vector compared to administration at 60 days gestation; (2) vector biodistribution was similar after administration by the i.p. or i.h. routes; and (3) gene expression analysis in transduced tissues showed the presence of mRNA transcripts that correlated with the level of gene transfer. These studies suggest that fetal gene transfer using the i.p. and i.h. routes results in prolonged transduction and expression of the transgene in multiple tissues.

Lentiviral vector gene transfer into fetal rhesus monkeys (Macaca mulatta): lung-targeting approaches.

We previously reported the efficiency of gene transfer in fetal monkeys using retroviral vectors and an intraperitoneal (IP) approach. Here, we explored intrapulmonary administration to determine whether gene transfer can be limited to the developing lung. The HIV-1-derived lentiviral vector (VSV-G pseudotyped; 1 x 10(7) infectious particles/fetus), using the enhanced green fluorescent protein (EGFP) as a reporter, was directly injected into fetal lung with ultrasound guidance (n=4; 55 or 70 days gestation; term 165+/-10 days). Fetuses were monitored sonographically, fetal/maternal blood samples collected during gestation, and four of four healthy newborns were delivered at term. All lung lobes were positive for the transgene (< or = 1%) when assessed by PCR, and transgene expression was observed by direct fluorescence microscopy and flow cytometry. The results of this study show the following: (1) successful gene transfer in fetal monkeys using an intrapulmonary approach; (2) less transduction of non-pulmonary tissues with gene transfer at 70 days gestation compared with 55 days gestation or use of an IP approach; (3) that the pulmonary epithelium was EGFP-positive by immunohistochemistry; and (4) no evidence of transplacental transport of vector sequences or antibody responses in the dams. The results of these investigations indicate the efficiency of fetal gene transfer by intrapulmonary delivery, and emphasize the importance of the fetal monkey as a preclinical model system for exploring in utero genetic treatment strategies for pulmonary disorders.

Rhesus monkey model for fetal gene transfer: studies with retroviral- based vector systems.

Many life-threatening conditions that can be diagnosed early in gestation may be treatable in utero using gene therapy. In order to determine in utero gene transfer efficiency and safety, studies were conducted with fetal rhesus monkeys as a model for the human. Included in these studies were Moloney murine leukemia virus (MLV)-based amphotropic retrovirus, vesicular stomatitis virus-G (VSV-G) pseudotyped MLV, and a VSV-G pseudotyped HIV-1-based vector, all expressing the enhanced green fluorescent protein (EGFP) as a reporter gene and driven by a cytomegalovirus-immediate early promoter (N = 16). Rhesus monkey fetuses were administered viral vector supernatant preparations by the intraperitoneal (ip) (N = 14) or intrahepatic (ih) (N = 2) routes via ultrasound guidance at 55 +/- 5 days gestation (late first trimester; term 165 +/- 10 days). Fetuses were monitored sonographically, specimens were collected prenatally and postnatally, and tissue harvests were performed at birth or 3 or 6 months postnatal age (3-10 months post-gene transfer). PCR analyses demonstrated that transduced cells were present at approximately 1.2% in peripheral blood mononuclear cells from fetuses administered amphotropic MLV, <0.5% in fetuses receiving MLV/VSV-G, and approximately 4.2% for the lentiviral vector, which decreased to 2% at birth. Hematopoietic progenitors showed that overall (mean of all time points assessed), approximately 25% of the collected colonies were positive for the EGFP transgene with the lentiviral vector, which was significantly greater than results achieved with the MLV-based vector systems (4-9%; P < or = 0.001-0.016). At necropsy, 0.001-10% of the total genomic DNA was positive for EGFP in most tissues for all groups. EGFP-positive fluorescent cells were found in cell suspensions of thymus, liver, spleen, lymph nodes, cerebral cortex, and bone marrow (0.5-6%). Overall, the results of these studies have shown: (1) healthy infants expressing vector sequences up to 10 months post-gene transfer, (2) fetal primate administration of retroviral vectors results in gene transfer to multiple organ systems, (3) the highest level of gene transfer to hematopoietic progenitors was observed with the lentiviral vector system, and (4) there was no evidence of transplacental transfer of vector sequences into the dams. The rhesus monkey is an important preclinical primate model system for exploring gene transfer approaches for future applications in humans.

Fetal rhesus monkey model of obstructive renal dysplasia.

BACKGROUND: Disorders of kidney development represent a major cause of renal failure and end-stage renal disease in the pediatric population. To understand further the prenatal pathogenesis of obstructive renal dysplasia, a fetal monkey model was developed using ultrasound-guided techniques. METHODS: Ureteropelvic obstruction (N = 13) was induced during the early or late second trimester by the injection of purified guluronic alginate spheres. All fetuses were monitored sonographically, and then fetal tissues were removed at varying time points during the second and third trimesters. RESULTS: There was no evidence of oligohydramnios during the course of gestation, and the obstructed kidneys were typically progressively smaller than the contralateral (nonobstructed) kidneys when monitored sonographically over time. Obstructed kidneys displayed most features of renal dysplasia, including numerous cortical cysts of various sizes derived predominantly from collecting ducts and glomeruli. Mesenchymal changes included expansion of both the cortical and medullary interstitium, as well as mesenchymal-myocyte transformation, expressed as pericystic and peritubular fibromuscular collar formation. An important feature of this model was the disruption of normal glomerular development and architecture, associated with significant podocyte apoptosis, evident as early as the prevascularized S-shaped nephron. As in other models, collecting duct cell apoptosis was apparent, particularly in areas of cyst formation and cellular atrophy. CONCLUSIONS: These results demonstrate the importance of this nonhuman primate model for exploring the pathophysiology of congenital obstructive uropathy and highlight the potential role of podocyte injury in determining long-term renal function associated with this condition.

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.

Tolerance induction post in utero stem cell transplantation.

The potential advantage of in utero HSC transplantation over a postnatal BMT is that early curative therapy could be given to an affected fetus, thus eliminating standard intensive immunosuppressive, marrow-ablative conditioning. It is apparent from studies in animals and humans that MHC-mismatched donor HSC of either fetal or adult origin can engraft in fetal recipients if the transplants are done sufficiently early in gestation. However, except for SCID, the percentage of donor pluripotent HSC that engraft is unacceptably low. We had hoped that for diseases such as thalassemia there would be a selective survival advantage for committed donor progenitor cells resulting in a high percentage of donor cell engraftment. At least based upon the experience in human fetuses with alpha- or beta-thalassemia, this has not been the case. Furthermore, for the majority of potential recipients of in utero HSC transplants, the marrow is non-defective, and the small percentage of pluripotent donor HSC that engraft would not be expected to selectively expand post-transplant. Our own results suggest that the non-defective fetal mouse and rhesus monkey are excellent models in which to study both stem cell engraftment, rejection, and tolerance induction. In our studies in non-defective mice with normal hematopoiesis, while the percentage of donor cells that are present is quite low, in only a small number of these animals were we able to induce permanent skin graft tolerance. Thus, while we found microchimerism in approximately 75% of recipients, less than 10% became tolerant. Even when we co-injected a large number of DC precursors, similar to what has been shown to induce tolerance to allogeneic liver, most of the animals failed to become tolerant to donor skin grafts. Interestingly, donor c-kit+ cells can be recruited with cytokines into the peripheral blood in engrafted mice, although these cells do not seem to be sufficient to induce tolerance to donor skin grafts, suggesting that the type (and location) of the engrafted donor cell plays a key role in tolerance induction. Our results in the fetal monkey model parallel those in the mouse, i.e., only a small number of donor cells engraft with limited tolerance induction. Interestingly, we found in our study of DC that GVHD was induced in those murine recipients of both allogeneic marrow and DC. It is likely that there were a sufficient number of mature DC in the preparation to facilitate a donor cytotoxic response towards the host. As a consequence there was also a significant increase in the percentage of donor cells that engrafted in the survivors. Future studies will focus on ways of blocking the graft vs host reaction while still maintaining the graft-promoting role of the donor T cell.

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.

Pharmacokinetics, pharmacodynamics, and safety of administering pegylated recombinant megakaryocyte growth and development factor to newborn rhesus monkeys.

Thrombocytopenia is common among sick neonates. Certain groups of thrombocytopenic adults respond favorably to the administration of recombinant thrombopoietin or to pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF), a recombinant human polypeptide that contains the receptor-binding N-terminal domain of thrombopoietin. The effectiveness and safety of such treatment in neonates, however, have not been reported. The purpose of the present study was to determine the biologic activity and safety of PEG-rHuMGDF administration to newborn rhesus monkeys. Eight monkeys were divided into four groups and treated subcutaneously with 0.00, 0.25, 1.00, or 2.50 microg/kg once daily for 7 d. Complete blood counts, serum chemistries, clotting panels, and MGDF levels were followed serially, and hematopoietic progenitor cell assays were performed on bone marrow aspirates before the first dose and again on d 8. Pharmacokinetic evaluations were performed on the animals that received the highest dose of PEG-rHuMGDF. All monkeys had normal growth during the study period, and all chemistries, clotting studies, and blood pressure measurements were normal. The peak serum MGDF concentration occurred at 3 h, and the half-life was 8.4 to 13.0 h. As in adult rhesus monkeys, platelet counts in the treated neonates began to rise on d 6, peaked on d 11, and returned to baseline by d 23. The two highest doses generated an 8- to 12-fold increase in platelets, whereas those treated with 0.25 microg/kg had a 6-fold increase. Other hematologic parameters measured were unaffected. Thus, newborn monkeys responded to doses of PEG-rHuMGDF that were similar to or smaller than (per kilogram body weight) those that are effective in adult animals and did so without obvious short-term toxicity.

Transabdominal ultrasonographic determination of fetal gender in the horse during mid-gestation.

Gender determination of the equine fetus using transabdominal ultrasonography was studied in 20 mares. One group of 10 research mares was scanned repeatedly every 2 weeks from 100 days gestation to parturition, while the second group of 10 client mares was subjected to echography once during mid-gestation. In males, the penis and/or prepuce was observed on 71 occasions from 102 days to 258 days gestation. On cross-sectional views, the male external genitalia had a round shape with parallel linear echogenic foci up to approximately 140 days gestation and then appeared triangular. Fetal testes were oval in shape in frontal view and had an homogeneous ultrasonographic appearance. Females were diagnosed on 23 occasions from 118 days to 227 days gestation based on the presence of the mammary glands and teats. Fetal ovaries appeared homogeneous with a characteristic circular echo from 100 days to 134 days gestation. Gender identifications (n = 98) based on the presence of the penis and/or prepuce in males and mammary glands and teats or fetal gonads in females were all correct, in agreement with the sex of the foals at birth. The optimal window of time was defined in both sexes as 100 to 220 days gestation. Thereafter, it was increasingly difficult to identify the anatomical structures cited above. Fetal sex was mainly determined using the transabdominal approach (87/98). However, the transrectal approach was useful in cases in which fetuses were either in posterior presentation or located very high in the mares abdomen. Good quality diagnostic scanners used typically in equine reproduction and equipped with a 5.0 MHz probe can be used for this procedure up to 160 days gestation, after which a 3.5 MHz transducer is often necessary due to increasing fetal size.

Endocrine biomarkers of early fetal loss in cynomolgus macaques (Macaca fascicularis) following exposure to dioxin.

This study examines the endocrine alterations associated with early fetal loss (EFL) induced by an environmental toxin, TCDD (2,3,7, 8-tetrachlorodibenzo-p-dioxin), in the cynomolgus macaque, a well-documented reproductive/developmental model for humans. Females were administered single doses of 1, 2, and 4 microgram/kg TCDD (n = 4 per dose group) on gestational day (GD) 12. Urinary estrogen metabolites (estrone conjugates) were monitored to establish the day of ovulation, and serum hormones (estradiol, progesterone, chorionic gonadotropin, relaxin) were measured to assess ovarian and placental endocrine status before and after treatment. EFL occurred between GDs 22 and 32 in 10 of the 12 animals treated with TCDD. The primary endocrine alterations associated with TCDD treatment were significant decreases in serum estradiol and bioactive chorionic gonadotropin concentrations (p < 0.02). Less pronounced decreases in serum progesterone (p = 0.10) and relaxin (p < 0.08) also followed TCDD treatment. In contrast, immunoreactive chorionic gonadotropin concentrations were not reduced by TCDD exposure at any level, indicating that TCDD targets specific components of the chorionic gonadotropin synthesis machinery within the trophoblast to alter the functional capacity of the hormone. These data demonstrate the value of endocrine biomarkers in identifying a toxic exposure to primate pregnancy many days before direct signs of reproductive toxicity were apparent. The increased EFL that occurred after exposure to TCDD might reflect a toxic response initially mediated via endocrine imbalance, leading to placental insufficiency, compromised embryonic circulation, and subsequent EFL.

Neuropathogenesis induced by rhesus cytomegalovirus in fetal rhesus monkeys (Macaca mulatta).

Rhesus cytomegalovirus (RhCMV) infection of rhesus macaques offers opportunities to analyze mechanisms of CMV pathogenesis in a primate species. Four fetal rhesus monkeys were inoculated intraperitoneally with RhCMV early in the second trimester, and pregnancies were terminated by hysterotomy during the third trimester. Three fetuses had evidence of severe CMV disease, including intrauterine growth restriction, ventriculomegaly, microcephaly, lissencephaly, and extensive degenerative changes of the cerebral parenchyma. Histopathologic examination revealed polymicrogyria, gliosis, leptomeningitis, periventricular calcifications, and inclusion-bearing cells. These results demonstrate that the developing macaque brain is susceptible to infection with RhCMV early in the second trimester and that intrauterine infection results in neuropathologic outcomes similar to those observed in humans congenitally infected with CMV.

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.

In utero hematopoietic stem cell transplants prolong survival of postnatal kidney transplantation in monkeys.

The authors hypothesized that in utero transplantation of T-cell-depleted paternal marrow into rhesus monkey fetuses would induce tolerance to postnatal kidney grafts from the marrow donor. T-cell-depleted paternal bone marrow was transplanted intraperitoneally into two female fetal rhesus monkeys at 61 +/- 1 days' gestation. Chimeric monkeys (n = 2) received kidney transplants from paternal donors. Control monkeys (n = 2) underwent kidney transplants without prior in utero stem cell transplants. Both chimeric monkeys demonstrated low level (<0.1% donor cells) engraftment in the bone marrow and peripheral blood using the polymerase chain reaction assay for the Y chromosome. The mixed lymphocyte reaction demonstrated hyporeactivity to the donor. Control animals demonstrated severe acute rejection and graft failure 1 week posttransplant. The first chimeric monkey had no significant clinical or sonographic evidence of renal failure until 7 weeks after the transplant. Biopsy findings showed mild rejection 1 week postoperatively, but rejection did not significantly progress until 5 weeks later. The second chimeric monkey had no significant clinical or sonographic changes for 4 weeks, but evidence of moderate rejection was seen on biopsy results. This monkey was given a 10-week course of immunosuppression, and had no clinical or sonographic renal deterioration, although biopsy results showed chronic rejection that was confirmed when electively euthanized 8 months later. Our data suggest that in utero transplantation of hematopoietic stem cells can increase the survival of a kidney allograft in the rhesus monkey.

Detection of Neospora from tissues of experimentally infected rhesus macaques by PCR and specific DNA probe hybridization.

Neospora is a newly recognized Toxoplasma-like cyst-forming coccidian parasite that causes abortion or congenital infections in naturally or experimentally infected animals. In this study, pregnant rhesus macaques were inoculated with culture-derived tachyzoites of a bovine Neospora isolate, and tissue samples from various major organs were collected from dams and fetuses for the detection of parasite DNA by using oligonucleotide primers COC-1 and COC-2 for PCR amplification of a conserved coccidial nuclear small-subunit rRNA gene sequence, and amplification products were confirmed by hybridization with a Neospora-specific DNA probe. PCR products were amplified from DNAs of different fetal monkey tissues, including brain, heart, lung, liver, spleen, skeletal muscle, skin, and placenta. In addition, Neospora DNA was amplified from the brain, heart, and lung tissues of infected rhesus macaque dams. The PCR and probe hybridization system may provide an effective method for the detection of Neospora infection in fetuses and dams from nonhuman primates and may be useful in determining the zoonotic potential of Neospora.

Pregnancy detection by ultrasound and chorionic gonadotropin during the peri-implantation period in the macaque (Macaca fascicularis).

The goal of these studies was to correlate sonographic evidence of pregnancy during the peri-implantation period with the timing of the rise in monkey chorionic gonadotropin (mCG) as measured with an enzyme-linked immunosorbent assay. Animals were time-mated at mid-cycle, and ultrasound examinations were performed on postovulation days 12-15 (n = 77). Pregnancy was sonographically identified in 48 of 77 animals (62.3%), of which 28 had correlative ultrasound/endocrine data collected. For these animals, blood samples were obtained on postovulation days 12-15 for mCG assay. Pregnancy was identified by ultrasound on postovulation days 12 (6/28; 21.4%), 13 (6/28; 21.4%), 14 (8/28; 28.6%) or 15 (8/28; 28.6%). Seven of the 28 (25.0%) were found to have mCG levels consistent with pregnancy (> or = 1 ng/ml) on the same day as ultrasound confirmation, 12 of 28 (42.9%) were sonographically detected as pregnant 1 (n = 6), 2 (n = 3) or 3 (n = 3) days earlier than by mCG, and nine of 28 (32.1%) were found to have elevated mCG levels 1 (n = 7), 2 (n = 1) or 3 (n = 1) days earlier than ultrasound confirmation of pregnancy. The results of these studies have demonstrated (1) the utility of anatomical and endocrine techniques for detecting pregnancy approximately 3 days after the onset of implantation, and (2) the variation in the timing of implantation and the rise in circulating mCG in individual animals.