Nonhuman Primate Tissues Research Articles

SRB Reproduction, Fertility and Development Award Lecture 2008. Regulation and manipulation of angiogenesis in the ovary and endometrium

The marked cyclical physiological angiogenesis in the developing follicle, corpus luteum and endometrium implies a critical role in health and disease. Our approach to understanding its regulation has been to localise and quantify the temporal changes in putative angiogenic factors, and their receptors, in human and non-human primate tissue and to use antagonists to dissect their role by specific inhibition at defined periods during the ovulatory cycle in non-human primates in vivo. The course of angiogenesis throughout the cycle and the cellular and molecular effects of inhibitory treatments have been investigated in the marmoset ovary and uterus, whereas consequences on pituitary-ovarian function have been monitored in macaques. Inhibition of vascular endothelial growth factor (VEGF) at the time of follicle recruitment or selection prevents endothelial cell proliferation, leading to inhibition of follicular development. VEGF inhibition during the early luteal phase prevents angiogenesis and restricts development of the luteal microvasculature. Inhibition of angiogenesis at all stages of the cycle leads to profound suppression of ovarian function. Even during the 'post-angiogenic' period of the luteal phase, inhibition of VEGF precipitates a suppression of progesterone secretion, pointing to additional roles for VEGF in the ovary. In the endometrium, oestrogen drives endometrial angiogenesis through VEGF. Thus, oestrogen can restore angiogenesis after ovariectomy, but not in the presence of VEGF inhibitors. These investigations enhance our understanding of the regulation of angiogenesis in the ovary and uterus and inform studies on conditions with abnormal vascularisation, such as polycystic ovary syndrome, endometriosis, uterine fibroids and menstrual dysfunction.

Endocrine-Related Resources from the National Institutes of Health

Endocrine-Related Resources from the National Institutes of Health

Xenotransplantation of solid organs in the pig-to-primate model

Xenotransplantation using pig organs could solve the significant increasing shortage of donor organs for allotransplantation. In the last two decades, major progress has been made in understanding the xenoimmunobiology of pig-to-nonhuman primate transplantation, and today we are close to clinical trials. The ability to genetically engineer pigs, such as human decay-accelerating factor (hDAF), CD46 (membrane cofactor protein), or α1,3-galactosyltransferase gene-knockout (GT-KO), has been a significant step toward the clinical application of xenotransplantation. Using GT-KO pigs and novel immunosuppressant agents, 2 to 6 months' survival of heterotopic heart xenotransplants has been achieved. In life-supporting kidney xenotransplantation, promising survival of close to 3 months has been achieved. However, liver and lung xenotransplantations do not have such encouraging survival as kidney and heart xenotransplantation. Although the introduction of hDAF and GT-KO pigs largely overcame hyperacute rejection, acute humoral xenograft rejection (AHXR) remains a challenge to be overcome if survival is to be increased. In several studies, when classical AHXR was prevented, thrombotic microangiopathy and coagulation dysregulation became more obvious, which make them another hurdle to be overcome. The initiating cause of failure of pig cardiac and renal xenografts may be antibody-mediated injury to the endothelium, leading to the development of microvascular thrombosis. Potential contributing factors toward the development of the thrombotic microangiopathy include: 1) the presence of preformed anti-non-Gal antibodies, 2) the development of very low levels of elicited antibodies to non-Gal antigens, 3) natural killer cell or macrophage activity, and 4) inherent coagulation dysregulation between pigs and primates. The breeding of pigs transgenic for an ‘anticoagulant’ or ‘anti-thrombotic’ gene, such as human tissue factor pathway inhibitor, hirudin, or CD39, or lacking the gene for the prothrombinase, fibrinogen-like protein-2, is anticipated to inhibit the change in the endothelium to a procoagulant state that takes place in the pig organ after transplantation. A further limitation for organ xenotransplantation is the potential for cross-species infection. As far as exogenous viruses are concerned, porcine cytomegalovirus has been detected in the tissues of recipient non-human primates, although no invasive disease was reported. Until today, no formal evidence has been presented from in vivo studies in non-human primates or from humans exposed to pig organs, tissues, or cells that porcine endogenous retroviruses infect primate cells. Xenotransplantation is a potential answer to the current organ shortage. Its future depends on; 1) further genetic modification of pigs, 2) the introduction of novel immunosuppressive agents that target the innate immune system and plasma cells, and 3) the development of clinically-applicable methods to induce donor-specific tolerance.

Endocrine-Related Resources from the National Institutes of Health

Endocrine-Related Resources from the National Institutes of Health

Endocrine-Related Resources from the National Institutes of Health

Endocrine-Related Resources from the National Institutes of Health

Endocrine-Related Resources from the National Institutes of Health

Endocrine-Related Resources from the National Institutes of Health

Endocrine-Related Resources from the National Institutes of Health

Endocrine-Related Resources from the National Institutes of Health

Pancreatic islet isolation variables in non-human primates (rhesus macaques)

Non-human primates (NHPs) are important preclinical models for pancreatic islet transplantation (PIT) because of their close phylogenetic and immunological relationship with humans. However, low availability of NHP tissue, long learning curves and prohibitive expenses constrain the consistency of isolated NHP islets for PIT studies. To advance preclinical studies, we attempted to identify key variables that consistently influence the quantity and quality of NHP islets. Seventy-two consecutive pancreatic islet isolations from rhesus macaques were reviewed retrospectively. A scaled down, semi-automated islet isolation method was used, and monkeys with streptozotocin-induced diabetes, weighing 3-7 kg, served as recipients for allotransplantation. We analysed the effects of 22 independent variables grouped as donor factors, surgical factors and isolation technique factors. Islet yields, success of isolation and transplantation results were used as quantitative and qualitative outcomes. In the multivariate analysis, variables that significantly affected islet yield were the type of monkey, pancreas preservation, enzyme lot and volume of enzyme delivered. The variables associated with successful isolation were the enzyme lot and volume delivered. The transplant result was correlated with pancreas preservation, enzyme lot, endotoxin levels and COBE collection method. Islet quantity and quality are highly variable between isolations. The data reviewed suggest that future NHP isolations should use bilayer preservation, infuse more than 80 ml of Liberase into the pancreas, collect non-fractioned tissue from the COBE, and strictly monitor for infection.

Endocrine-Related Resources from the National Institutes of Health

Endocrine-Related Resources from the National Institutes of Health

Endocrine-Related Resources from the National Institutes of Health

Endocrine-Related Resources from the National Institutes of Health

Endocrine-Related Resources from the National Institutes of Health

Endocrine-Related Resources from the National Institutes of Health

Complete Spermatogenesis in Orthotopic But Not in Ectopic Transplants of Autologously Grafted Marmoset Testicular Tissue

Testicular grafting has the potential to become a method to preserve fertility in prepubertal boys undergoing cancer treatment. The possibility of successful germ cell maturation after autologous grafting should be proven preclinically in a nonhuman primate model. Therefore, in two experiments, we analyzed the potential of autologous testicular grafting in the marmoset model. A first experiment in immature and adult hemi-castrated monkeys addressed the question of whether full spermatogenesis in an ectopic graft could be achieved under a relatively normal endocrine milieu and whether the donor's age is of influence. A second experiment in castrated immature animals examined whether the transplantation site [ectopic (back skin) or orthotopic (scrotum)] influences spermatogenic progress and whether cryopreserved tissue can be successfully transplanted. Grafts were analyzed by histology, immunohistochemistry, and morphometry. Bioactive chorionic gonadotropin and serum testosterone were measured. In the adults, ectopic grafts degenerated, whereas in the immature animals, grafts survived at the spermatogonial level. In the castrates, none of the cryopreserved grafts survived, ectopic grafts were meiotically arrested, but orthotopic transplants completed spermatogenesis. Androgen and bioactive chorionic gonadotropin levels were not decisive for graft development. When ectopic and orthotopic transplantation sites were compared, the scrotum has a substantially lower temperature. Thus, the higher temperature at the ectopic transplantation site may contribute to spermatogenic arrest. Autologous grafting of nonhuman primate testicular tissues can result in complete spermatogenesis. Our findings indicate that transplantation site and developmental age of the tissue play a role more important than the endocrine milieu.

Biochemical, Pathological, and Skeletal Improvement of Mucopolysaccharidosis VI After Gene Transfer to Liver but Not to Muscle

Mucopolysaccharidosis VI (MPS VI) is caused by deficient activity of arylsulfatase B (ARSB), resulting in intralysosomal storage of dermatan sulfate (DS) and multisystem disease without central nervous system involvement. After gene transfer, muscle or liver can theoretically be converted into factories for systemic ARSB secretion, leading to uptake by non-transduced cells. We have injected newborn MPS VI rats and cats with adeno-associated viral (AAV) vectors expressing ARSB under the control of liver-specific, muscle-specific, or universally active promoters. After systemic or intramuscular (IM) administration of AAV, therapeutic levels of circulating ARSB are achieved, resulting in skeletal improvements and significant decrease in glycosaminoglycan (GAG) storage, inflammation and apoptosis (despite a neutralizing immune response to ARSB in MPS VI rats). In addition, we have observed wide-spread dissemination of vector after IM AAV administration. This results in secretion of therapeutic levels of ARSB when the universally active cytomegalovirus (CMV) but not the muscle-specific muscle creatine kinase (MCK) promoter is used, suggesting that transduction of extramuscular sites rather than enzyme secretion from muscle occurs after muscle ARSB gene transfer. We conclude that AAV-mediated expression of ARSB from liver represents a feasible therapeutic strategy for MPS VI, potentially avoiding multiple infusions of costly recombinant enzyme associated with enzyme replacement therapy.

Effect of cold storage and cryopreservation of immature non-human primate testicular tissue on spermatogonial stem cell potential in xenografts

Effect of cold storage and cryopreservation of immature non-human primate testicular tissue on spermatogonial stem cell potential in xenografts

Endocrine-Related Resources from the National Institutes of Health

Endocrine-Related Resources from the National Institutes of Health

Endocrine-Related Resources from the National Institutes of Health

Endocrine-Related Resources from the National Institutes of Health

Metal Particles and Extrapulmonary Transport: Oberdörster and Elder Respond

We appreciate the opportunity to address the points raised by Ghio and Bennett in their letter. The pH of our cell-free dissolution studies (Elder et al. 2006) was appropriate because the nasal cavity surface is neutral and does not have airway macrophages and the phagolysosomal pH of nasal epithelial cells is neutral (Johnson 1994). Acidic pH, as in the phagolysosome of alveolar macrophages, dissolves manganese oxide, resulting in increased levels of blood-borne non-particulate Mn. Our neutral pH buffer dissolved only 1.5% of Mn oxide nanoparticles in 24 hr. The dissolution buffer was not “normal saline,” as stated by Ghio and Bennett; it was physiologic saline, a “simulated lung fluid” used for decades in studies of man-made fiber dissolution (Potter and Mattson 1991). It was an oversight on our part to omit the exact composition from our article (Elder et al. 2006), which includes citrate (model organic acid) and glycine (model protein component). Citrate is a stronger metal chelator than glycine. If only soluble Mn translocates, the time required for solubilization would significantly retard the increase of Mn in the olfactory bulb of administered Mn oxide nanoparticles. Instead, we found no difference between the two forms of Mn when we directly compared the translocation rate of the Mn oxide nanoparticles to soluble Mn chloride (Elder et al. 2006), indicating a direct uptake of the Mn oxide nanoparticles by olfactory neuronal structures and subsequent translocation. It is conceivable that subsequent dissolution in the olfactory system occurs. The rapidity of solid nanoparticle transport along neuronal axons is, indeed, remarkable (~ 2.5 mm/hr), as demonstrated earlier by the arrival in the olfactory bulb of 50 nm gold particles within 30 min after intranasal instillation. This and other studies with gold nanoparticles using transmission electron microscopy detection (reviewed by Oberdorster et al. 2005) demonstrate unequivocally that some metal particles are indeed appropriate for demonstrating solid particle transport across epithelial barriers, refuting the absolute statement in the title of Ghio and Bennett’s letter. Ghio and Bennett suggest that a carbon-based particle would be appropriate for studying ultrafine particle transport and translocation. This is true for elemental and organic carbon only if insoluble in vivo. Indeed, study with inhaled ultrafine elemental carbon particles (13C) confirmed their translocation to the olfactory bulb of rats (Oberdorster et al. 2004). In contrast, labeled elemental carbon is problematic, as pointed out by Ghio and Bennett regarding Technegas (99mTc labeled-ultrafine carbon). Recent studies using inhaled Technegas (Mills et al. 2006; Wiebert et al. 2006) showed no translocation in humans, contradicting earlier work (Nemmar et al. 2002). Both leaching of the soluble radiolabel and the inability of the g-camera to detect small amounts (£ 1%) of the deposited dose in extrapulmonary organs are significant limitations with this noninvasive technique, resulting in misinterpretions suggesting either significant particle translocation or lack thereof. Ghio and Bennett cite LeFevre et al. (1982), apparently as evidence that inhaled carbon-based particles do not undergo extrapulmonary transport. However, LeFevre et al. interpreted their findings of high black pigment scores in liver and spleen of coal miners differently—namely, as migration of coal dust in the pneumoconiotic lung into pulmonary lymphatics and then to the systemic circulation, and also as migration of coal mine dust–laden macrophages through the walls of pulmonary blood vessels. Whatever the mechanism, their findings clearly indicate extrapulmonary transport. Heavy silica inhalation exposure has also been found to result in particle accumulation in liver and other extrapulmonary tissues in humans and nonhuman primates (Carmichael et al. 1980; Rosenbruch 1990; Slavin et al. 1985). Ghio and Bennett further state that Decades of research have provided no evidence of an extrapulmonary transport (including via olfactory neuronal pathways) of particles associated with cigarette smoking. How many investigators have tried to find cigarette smoke particles in extrapulmonary tissues? We are aware of only one study in rats in which a 25-min inhalation exposure to 14C cigarette smoke resulted in 0.24–0.83% of retained 14C in the liver within 15 min after the short-term exposure (Chen et al. 1989). Does this indicate extrapulmonary particle transport? Possibly yes. We conclude that the physicochemical characteristics of a nanoparticle—whether metal or not—and the physiologic milieu at the site of deposition in the respiratory tract determine whether extrapulmonary translocation occurs as particle or as solute. A prerequisite for noninvasively measuring this is that the detection method has sufficient sensitivity for identifying the analyte at expected low translocation rates. Regarding our nasal translocation study in rats (Elder et al. 2006), we conclude that inhaled Mn oxide nanoparticles are taken up by the nasal olfactory neuronal pathway as solid particles rather than being slowly dissolved first at neutral pH. For the alveolar region where dissolution is expected to be more rapid, this does not apply.

A new test to measure the strength of visual exploration in common marmoset

Profiles of recombinant adeno-associated virus (rAAV)-mediated transduction show interspecies differences for each AAV serotype. Robust long-term transgene expression is generally observed in rodents, whereas insufficient transduction is seen in animals with more advanced immune systems. Non-human primates, including the common marmoset, could provide appropriate models for neuromuscular diseases because of their higher brain functions and physiological resemblance to humans. Strategies to induce pathologies in the neuromuscular tissues of non-human primates by rAAV-mediated transduction are promising; however, transgene expression patterns with rAAV transduction have not been elucidated in marmosets. In this study, transduction of adult marmoset skeletal muscle with rAAV9 led to robust and persistent enhanced green fluorescent protein (EGFP) expression that was independent of the muscle fiber type, although lymphocyte infiltration was recognized. Systemic rAAV injection into pregnant marmosets led to transplacental fetal transduction. Surprisingly, the intraperitoneal injection of rAAV1 and rAAV9 into the neonatal marmoset resulted in systemic transduction and persistent transgene expression without lymphocyte infiltration. Skeletal and cardiac muscle were effectively transduced with rAAV1 and rAAV9, respectively. Interestingly, rAAV9 transduction led to intense EGFP signaling in the axons of the corpus callosum. These transduction protocols with rAAV will be useful for investigating gene functions in the neuromuscular tissues and developing gene therapy strategies.

Effect of cold storage and cryopreservation of immature non-human primate testicular tissue on spermatogonial stem cell potential in xenografts

Successful cryopreservation of gonadal tissue is an important factor in guaranteeing the fertility preservation via germ cell or testicular tissue transplantation. The aim of this study was to evaluate the effects of cooling and cryopreservation on spermatogonial stem cell survival and function of immature non-human primate testicular tissue xenografted to nude mice. Group 1 (control group) received subcutaneous grafts of fresh immature rhesus monkey testes. The treatment groups received grafts after 24 h cooling in ice-cold medium (Group 2), after 24 h of cryopreservation without cryoprotectant (Group 3), with ethylene glycol (Group 4: 1.4 M) or with dimethylsulphoxide (DMSO) (group 5: 1.4 M; group 6: 0.7 M), using cooling rates of 0.5 degrees C/min. The graft number, weight and histology were examined 3-5 months later. After xenografting, grafts from fresh and cooled tissue showed good survival and spermatogenic induction to spermatocytes. Cryopreservation in 1.4 M DMSO also allowed grafts to initiate spermatogenesis. In contrast, 0.7 M DMSO and ethylene glycol showed inferior protection. Our observations suggest that cryopreservation of immature primate testis is a feasible approach to maintain spermatogonial stem cells and may serve as a promising tool for fertility preservation of prepubertal boys. The possibility to delay the transplantation of cooled samples suggests an option for clinical centralization of testicular tissue cryopreservation.

Xenotransplantation – ethics and regulation

Healthcare workers, researchers, and policy makers find themselves in challenging times, being required to make (often prompt) decisions in the face of novel ethical dilemmas. Moral theory may assist with decision-making in complex areas of medicine such as the xenotransplantation of insulin-producing cells to reverse diabetes. Moral theory attempts to capture intuitions about whether an action is right or wrong and it may gives us a deeper understanding of moral claims as it gives us an account as to why an action is good or bad. We may opt to choose different ethical frameworks such as consequentialism, principlism or a duty-based approach, to assist with deciding a certain action in a particular scenario. The xenotransplantation of porcine insulin-producing cells is a potential solution to the shortage of human tissue. It is crucial for the advancement of xenotransplantation that the benefits to society outweigh the risks and whilst there is enormous potential to assist mankind, there are unique bioethical challenges and dilemmas. New treatment strategies should be regulated appropriately to minimize risks to patients, their relatives and the wider community. There are specific regulations (such as those developed by the FDA) that provide a robust framework for the safe implementation of xenotransplantation and more generally applicable ones such as the Declaration of Helsinki. Xenotransplantation is either morally acceptable or not to an individual. Patients must ultimately decide as to whether they would accept a certain form of treatment or not, a decision which would take into account their religious beliefs and categorical objections. There is no point in developing a treatment option unless there is good patient support. To that end, a study was conducted to ascertain the acceptance of tissue from different animal sources, by diabetic patients in England and Australia. Interviews were conducted to ensure that patients' views were reflected in their answers. There were 316 English and 195 Australian questionnaire respondents and the vast majority would accept human (85%, 94%), porcine (68%, 85%) or non-human primate (67%, 82%) tissue, respectively. This study suggests that a large proportion of diabetic patients may accept a xenotransplantation product.