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.

Improved collection and developmental competence of immature macaque oocytes.

Methods previously described to aspirate immature oocytes from ovaries of macaques result in approximately half the oocytes being stripped of cumulus cells. Here, we describe modifications of the needle aspiration assembly that yield much higher percentages of cumulus-intact oocytes when used with an ultrasound-guided method for oocyte recovery in monkeys. Sealing of the needle assembly appears to stabilize vacuum pressure at the needle tip and prevents air from entering the tubing. Reduction of the vacuum pressure from -100 to -20 kPa resulted in a significant decrease of denuded oocytes from over 50% to fewer than 10%. This was accompanied by a significant increase in the percentage of oocytes that developed into blastocysts after in vitro fertilization. Reduction of the aspiration pressure below -20 kPa significantly reduced the total number of oocytes recovered. We concluded that these modifications represent the best compromise to collect the largest number of cumulus-intact oocyte complexes from macaques.

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.

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.

Role of hypothalamic-pituitary axis in EGF action on maturation of adrenal gland in fetal rhesus monkey in vivo.

We determined the route of action of epidermal growth factor (EGF) [intraperitoneal (IP) versus intraamniotic administration] on adrenal development and whether its effects are mediated via the fetal hypothalamic-pituitary axis in the fetal rhesus monkey in vivo. EGF (40 microg) was administered IP (n = 9) or intraamniotic (n = 6) at 121, 123, 125, and 127 d gestation (term, approximately 165 +/- 10 d gestation). In addition, a competitive corticotropin-releasing factor antagonist ([D-phenylalanine(12), Norleucine(21,38)] corticotropin-releasing factor(12-41) to block fetal pituitary ACTH secretion; 400 microg IP) and metyrapone (11beta-hydroxylase inhibitor to block adrenal cortisol synthesis; 15 mg IP and 15 mg intraamniotic) were administered, in combination with EGF (EGF+BLOCK; 40 microg IP; n = 4 fetuses). Control fetuses (n = 6) received saline injections in an equivalent volume. On gestational d 128, a hysterotomy was performed, and fetal adrenals were collected for morphometric analyses and immunocytochemical localization of 3beta -hydroxysteroid dehydrogenase (3betaHSD) and cytochrome P-450 11beta -hydroxylase/aldosynthase. Definitive zone (DZ) width and cortical width of 3betaHSD staining were significantly greater (p < 0.05) in the EGF IP-treated fetuses compared with controls and EGF+BLOCK. With EGF IP, 3betaHSD was increased in the DZ and induced extensively in the transitional zone of the fetal adrenal cortex, and cytochrome P-450 11beta-hydroxylase/aldosynthase immunoreactivity was induced to detectable levels in the DZ. The administration of EGF+BLOCK inhibited the expression of 3betaHSD in the transitional zone, but 3betaHSD expression was still increased in the DZ and cytochrome P-450 11beta-hydroxylase/aldosynthase immunoreactivity was induced in the DZ. EGF intraamniotic administration had no significant effect on the width of the DZ or cortical width of 3betaHSD staining compared with controls. These data suggest that EGF acts via the hypothalamic-pituitary axis to modulate adrenal cortical growth and functional maturation of the transitional zone (the putative zona fasciculata), whereas EGF can act independently of the hypothalamic-pituitary axis to stimulate functional maturation of the DZ (the putative zona glomerulosa).

IGF-II and IGF binding protein (IGFBP-1, IGFBP-3) gene expression in fetal rhesus monkey tissues during the second and third trimesters.

The IGF system is a key modulator of somatic fetal growth. Studies with human fetal tissues have shown a specific spatial and temporal pattern of expression of IGF and IGF binding protein (IGFBP) mRNAs, but have been limited to defined periods during gestation (i.e. 8-20 wk gestation) because of tissue availability. To fully assess the role of these peptides in the primate growth process, a longitudinal study was conducted that focused on the expression of IGF-II and IGFBP-1 and IGFBP-3 genes in the rhesus monkey (Macaca mulatta). Liver, kidney, brain, and lung were collected from rhesus monkey fetuses approximately every 2 wk from 65 (early second trimester) through 150 d gestation (term 165 +/- 10 d) (n = 50), then processed for in situ hybridization using radiolabeled human cDNAs. IGF-II mRNA was abundantly expressed in fetal kidney (maturing glomerulus, supporting mesenchyme, cells of the developing nephrons), liver (hepatocytes), cerebral cortex (choroid plexus, capillaries), and lung (blood vessels, connective tissues, lamina propria, cartilage framework). IGFBP-1 was expressed only in the hepatocytes and IGFBP-3 mRNA was modestly expressed within the kidney (developing nephrons, collecting system mesenchyme), and liver (hepatocytes). These studies have shown that (1) IGF-II, IGFBP-1, and IGFBP-3 are expressed in specific cell types of the fetal monkey indicating a paracrine/autocrine role during development; (2) changes in IGF-II and IGFBP mRNA expression occur with advancing gestation; and (3) fetal monkey tissues express IGF-II and IGFBPs in a similar manner when compared with the human fetus.

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.

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.

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.

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.

Recombinant human gonadotropins for macaque superovulation: repeated stimulations and post-treatment pregnancies.

This report summarizes data from the superovulation and ultrasound-guided follicular aspiration of 40 female rhesus monkeys (Macaca mulatta) with recombinant human gonadotropins. Of the animals treated, 12 were stimulated for only one cycle, either because of a poor response to the hormones or due to ectopic ovarian position precluding ease of access via ultrasound. The majority of animals were stimulated for a minimum of 3 cycles and 3 females continued to respond for a minimum of 8 and a maximum of 10 cycles. For those animals with repeated stimulation cycles, the number of follicles developed during each of the stimulation protocols remained relatively comparable. Of the animals mated since cessation of treatment, 70% conceived. There was no difference between the conception rate in this subset of animals and the rest of the macaque breeding colony. These data indicate that participation in these studies does not impact on the reproductive potential of female rhesus monkeys.

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.

Evaluation of equine fetal growth from day 100 of gestation to parturition by ultrasonography.

Transrectal and transabdominal ultrasonography were performed on normal pregnant mares (n=10) at 2 week intervals from day 100 of gestation to parturition to evaluate fetal growth. Several fetal anatomical regions (head, eye, aorta, abdomen, rib, gonad, kidney and femur) were imaged and measured using standardized scan plans. The results of these analyses indicate that all of the biometric parameters correlate strongly with the day of gestation. Growth charts were developed, which demonstrate that the following variables have linear relationships with the day of gestation on which they were measured: aortic systolic diameter, biparietal diameter, approximate eye volume, femur length and kidney cross-sectional area. The linear regression equation across days was developed for aortic, systolic and biparietal diameter, approximate eye volume, femur length and kidney cross-sectional area, thus allowing assessment of normal equine fetal development after day 100 of gestation. This non-invasive method can be used to estimate fetal age if mating or ovulation dates are unknown, provided the fetus is developing normally. The use of transrectal and transabdominal ultrasonography, as well as different probe frequencies (5.0 or 3.5 MHz), to measure different biometric parameters during gestation is reported.