Use of Nonhuman Primates in Research in North America

In North America, the biomedical research community faces social and economic challenges to nonhuman primate (NHP) importation that could reduce the number of NHP available for research needs. The effect of such limitations on specific biomedical research topics is unknown. The Association of Primate Veterinarians (APV), with assistance from the Centers for Disease Control and Prevention, developed a survey regarding biomedical research involving NHP in the United States and Canada. The survey sought to determine the number and species of NHP maintained at APV members’ facilities, current uses of NHP to identify the types of biomedical research that rely on imported animals, and members’ perceived trends in NHP research. Of the 149 members contacted, 33 (22%) replied, representing diverse facility sizes and types. Cynomolgus and rhesus macaques were the most common species housed at responding institutions and comprised the majority of newly acquired and imported NHP. The most common uses for NHP included pharmaceutical research and development and neuroscience, neurology, or neuromuscular disease research. Preclinical safety testing and cancer research projects usually involved imported NHP, whereas research on aging or degenerative disease, reproduction or reproductive disease, and organ or tissue transplantation typically used domestic-bred NHP. The current results improve our understanding of the research uses for imported NHP in North America and may facilitate estimating the potential effect of any future changes in NHP accessibility for research purposes. Ensuring that sufficient NHP are available for critical biomedical research remains a pressing concern for the biomedical research community in North America.Abbreviation: APV, Association of Primate Veterinarians; CDC, Centers for Disease Control and Prevention; NHP, nonhuman primateNonhuman primates (NHP) remain key animal models for specific types of biomedical research because of their close phylogenetic relationships and physiologic similarities to humans.^1,8,10,12,18 However, the use of NHP in research has become increasingly controversial such that ensuring a stable supply of NHP to meet biomedical research needs continues to be a pressing concern.^4,16,23In fiscal year 2010, US research facilities housed more than 70,000 NHP for use in biomedical research.²¹ Although many North American facilities rely on domestic NHP breeding colonies for research needs, they also import purpose-bred NHP, primarily from Asia, to supplement domestic breeding. NHP importation into the United States peaked in 2008 at just over 25,000 animals but has experienced a small but steady decline each subsequent year.¹⁴ Many social, economic, and scientific factors might be contributing to this decrease, for example, development of alternatives to replace NHP models. One factor of great concern to researchers is increasing resistance among airlines to transport live NHP.^17,23Only a few reports describe the types of NHP species used and the research areas in which this model predominates.^3,9,20 Previous retrospective literature reviews provide valuable baseline information about NHP research activities for some species or geographic locations; however, these studies do not identify NHP procurement sources. Therefore, published literature does not identify which types of biomedical research are most reliant on imported NHP nor does it assist with understanding how changes in importation would affect the numbers of NHP available for specific biomedical research activities. A cross-sectional, survey-based approach provides more direct access to information about species use, NHP sources, and the types of studies that are most reliant on NHP importation.The Association of Primate Veterinarians (APV), with technical support from the Division of Global Migration and Quarantine at the US Centers for Disease Control and Prevention (CDC), collaboratively surveyed APV''s membership regarding biomedical research activities involving NHP at institutions in the United States and Canada. The goal of this survey was to document the number and species of imported NHP maintained at research facilities served by APV members, to understand the types of biomedical research that are particularly reliant on imported NHP, and to gain insight into practicing primate veterinarians’ perceptions of NHP research trends.     

Neurocardiac Responses to Vagoafferent Electrostimulation in Humans

To determine if cardiac vagal tone is enhanced by vagal electrostimulation (VES), we examined the heart rate autospectrum (HRA) in eight patients with implanted stimulators for complex partial seizures. In four patients the VES was activated at 30 Hz and 500-msec pulse (HiStim group) compared to 2 Hz and 130-msec pulse for the LoStim group (n = 4). Continuous ECG and respiratory waveforms were recorded for 45 minutes every 8 hours (7–8 AM; 3–4 PM 11–12 PM) during resting supine wakeful epochs both before and 15 days after surgical implantation. From the HRA cardiac sympathovagal balance was expressed as the ratio of the low frequency (LF) power to the high frequency (HF) power. RESULTS: There were no presurgical differences between the groups in heart rate, its variance, or the energies contained in any autospectral band. The LoStim group showed no significant change in heart rate (HR), HF peak power, or LF:HF ratios during 2 weeks of VES. Conversely, in the HiStim group, the LF:HF peak power ratio fan expression of sympathetic dominance) decreased from 2.5 ± 1.5 preimplant to 1.5 ± 0.49 (P < 0.02) with VES. During VES there was a significantly higher HF power in the HiStim compared to LoStim group. No diurnal variations in HRA values were seen for either group. CONCLUSIONS: (1) A relationship exists between selective vagal nerve electrostimulation and the HRA; and (2) high stimulation frequency of the vagus nerve in man is associated with sustained augmentation of cardiac vagal tone throughout a 24-hour cycle.     

Efficient Gene Delivery and Selective Transduction of Glial Cells in the Mammalian Brain by AAV Serotypes Isolated From Nonhuman Primates

Adeno-associated viral (AAV) vectors have become the primary delivery agent for somatic gene transfer into the central nervous system (CNS). To date, AAV-mediated gene delivery to the CNS is based on serotypes 1–9, with efficient gene transfer to neurons only—selective and widespread transduction of glial cells have not been observed. Recently, additional endogenous AAVs have been isolated from nonhuman primate tissues. In this study, transduction obtained with AAV serotypes bb2, cy5, rh20, rh39, and rh43 was compared to that obtained with AAV8, another nonhuman primate isolate previously shown to perform well in mammalian brain. Titer-matched vectors encoding the enhanced green fluorescent protein (EGFP) reporter, driven by the constitutive CAG promoter, were injected into the hippocampus, striatum, or substantia nigra (SN) of adult rats. More widespread neuronal transduction was observed following infusion of cy5, rh20, and rh39 than observed with AAV8. Of interest, preferential transduction of astrocytes was observed with rh43. To optimize glial transduction, vector stocks driven by cell-specific promoters were generated—widespread and targeted transduction of astrocytes and oligodendrocytes was observed using rh43 and AAV8, driven by the glial fibrillary acidic protein (GFAP) and myelin basic protein (MBP) promoters, expanding the utility of AAV for modeling and treating diseases involving glial cell pathology.     

Linezolid Activity Compared to Those of Selected Macrolides and Other Agents against Aerobic and Anaerobic Pathogens Isolated from Soft Tissue Bite Infections in Humans

Linezolid was tested against 420 aerobes and anaerobes, including 148 Pasteurella isolates, by an agar dilution method. Linezolid was active against all Pasteurella multocida subsp. multocida and P. multocida subsp. septica isolates and most Pasteurella canis, Pasteurella dagmatis, and Pasteurella stomatis isolates. The MIC was 相似文献   

Emergence of H7N9 Influenza A Virus Resistant to Neuraminidase Inhibitors in Nonhuman Primates

The number of patients infected with H7N9 influenza virus has been increasing since 2013. We examined the efficacy of neuraminidase (NA) inhibitors and the efficacy of a vaccine against an H7N9 influenza virus, A/Anhui/1/2013 (H7N9), isolated from a patient in a cynomolgus macaque model. NA inhibitors (oseltamivir and peramivir) barely reduced the total virus amount because of the emergence of resistant variants with R289K or I219T in NA [residues 289 and 219 in N9 of A/Anhui/1/2013 (H7N9) correspond to 292 and 222 in N2, respectively] in three of the six treated macaques, whereas subcutaneous immunization of an inactivated vaccine derived from A/duck/Mongolia/119/2008 (H7N9) prevented propagation of A/Anhui/1/2013 (H7N9) in all vaccinated macaques. The percentage of macaques in which variant H7N9 viruses with low sensitivity to the NA inhibitors were detected was much higher than that of macaques in which variant H5N1 highly pathogenic influenza virus was detected after treatment with one of the NA inhibitors in our previous study. The virus with R289K in NA was reported in samples from human patients, whereas that with I219T in NA was identified for the first time in this study using macaques, though no variant H7N9 virus was reported in previous studies using mice. Therefore, the macaque model enables prediction of the frequency of emerging H7N9 virus resistant to NA inhibitors in vivo. Since H7N9 strains resistant to NA inhibitors might easily emerge compared to other influenza viruses, monitoring of the emergence of variants is required during treatment of H7N9 influenza virus infection with NA inhibitors.     

Neonatal Systemic AAV Induces Tolerance to CNS Gene Therapy in MPS I Dogs and Nonhuman Primates

The potential host immune response to a nonself protein poses a fundamental challenge for gene therapies targeting recessive diseases. We demonstrate in both dogs and nonhuman primates that liver-directed gene transfer using an adeno-associated virus (AAV) vector in neonates induces a persistent state of immunological tolerance to the transgene product, substantially improving the efficacy of subsequent vector administration targeting the central nervous system (CNS). We applied this approach to a canine model of mucopolysaccharidosis type I (MPS I), a progressive neuropathic lysosomal storage disease caused by deficient activity of the enzyme α-l-iduronidase (IDUA). MPS I dogs treated systemically in the first week of life with a vector expressing canine IDUA did not develop antibodies against the enzyme and exhibited robust expression in the CNS upon intrathecal AAV delivery at 1 month of age, resulting in complete correction of brain storage lesions. Newborn rhesus monkeys treated systemically with AAV vector expressing human IDUA developed tolerance to the transgene, resulting in high cerebrospinal fluid (CSF) IDUA expression and no antibody induction after subsequent CNS gene therapy. These findings suggest that inducing tolerance to the transgene product during a critical period in immunological development can improve the efficacy and safety of gene therapy.     

Persistent Expression of FLAG-tagged Micro dystrophin in Nonhuman Primates Following Intramuscular and Vascular Delivery

Animal models for Duchenne muscular dystrophy (DMD) have species limitations related to assessing function, immune response, and distribution of micro- or mini-dystrophins. Nonhuman primates (NHPs) provide the ideal model to optimize vector delivery across a vascular barrier and provide accurate dose estimates for widespread transduction. To address vascular delivery and dosing in rhesus macaques, we have generated a fusion construct that encodes an eight amino-acid FLAG epitope at the C-terminus of micro-dystrophin to facilitate translational studies targeting DMD. Intramuscular (IM) injection of AAV8.MCK.micro-dys.FLAG in the tibialis anterior (TA) of macaques demonstrated robust gene expression, with muscle transduction (50–79%) persisting for up to 5 months. Success by IM injection was followed by targeted vascular delivery studies using a fluoroscopy-guided catheter threaded through the femoral artery. Three months after gene transfer, >80% of muscle fibers showed gene expression in the targeted muscle. No cellular immune response to AAV8 capsid, micro-dystrophin, or the FLAG tag was detected by interferon-γ (IFN-γ) enzyme-linked immunosorbent spot (ELISpot) at any time point with either route. In summary, an epitope-tagged micro-dystrophin cassette enhances the ability to evaluate site-specific localization and distribution of gene expression in the NHP in preparation for vascular delivery clinical trials.     

Differential Transduction Following Basal Ganglia Administration of Distinct Pseudotyped AAV Capsid Serotypes in Nonhuman Primates

We examined the transduction efficiency of different adeno-associated virus (AAV) capsid serotypes encoding for green fluorescent protein (GFP) flanked by AAV2 inverted terminal repeats in the nonhuman primate basal ganglia as a prelude to translational studies, as well as clinical trials in patients with Parkinson''s disease (PD). Six intact young adult cynomolgus monkeys received a single 10 µl injection of AAV2/1-GFP, AAV2/5-GFP, or AAV2/8-GFP pseudotyped vectors into the caudate nucleus and putamen bilaterally in a pattern that resulted in each capsid serotype being injected into at least four striatal sites. GFP immunohistochemistry revealed excellent transduction rates for each AAV pseudotype. Stereological estimates of GFP⁺ cells within the striatum revealed that AAV2/5-GFP transduces significantly higher number of cells than AAV2/8-GFP (P < 0.05) and there was no significant difference between AAV2/5-GFP and AAV2/1-GFP (P = 0.348). Consistent with this result, Cavalieri estimates revealed that AAV2/5-GFP resulted in a significantly larger transduction volume than AAV2/8-GFP (P < 0.05). Each pseudotype transduced striatal neurons effectively [>95% GFP⁺ cells colocalized neuron-specific nuclear protein (NeuN)]. The current data suggest that AAV2/5 and AAV2/1 are superior to AAV2/8 for gene delivery to the nonhuman primate striatum and therefore better candidates for therapeutic applications targeting this structure.     

Refinement of a Protocol for the Induction of Lactation in Nonpregnant Nonhuman Primates by Using Exogenous Hormone Treatment

Obtaining sufficient quantities of milk from NHP is necessary for pharmacologic and immunologic studies required for the development and safety assessment of drugs and vaccines to be used in the maternal–infant setting. We previously induced lactation in nonpregnant female rhesus macaques (RM, Macaca mulatta) and African green monkeys (AGM, Chlorocebus sabaeus) for studies of immune responses in milk, but the volume collected was variable. To improve lactation induction protocols for nonbreeding nonhuman primates, we investigated serum hormone levels and collection protocols in AGM and RM. Here, we correlated milk volume with serum levels of endogenous and administered hormones: estradiol, prolactin, progesterone, and medroxyprogesterone in RM and AGM. We also investigated whether age, parity or the timing of milk collections were associated with the volume of milk collected from the AGM and RM in which lactation was induced by using exogenous hormones. We found an inverse correlation with serum estradiol and milk volume in the RM but no significant correlation between milk volumes and the remaining serum hormone levels in the induced RM or AGM. In addition, HIL AGM had higher peak estradiol levels than did naturally lactating AGM. A revised estradiol-sparing protocol increased milk volumes in the AGM. In addition, milk volume in RM was greater in the morning than the afternoon. In conclusion, we have refined a lactation induction protocol in nonpregnant primates, which is a needed alternative to using nursing primates for the assessment of drug levels and immune responses in milk.Abbreviations: AGM, African green monkey; AGM1, first AGM lactation induction; AGM2, second AGM lactation induction; RM, rhesus macaque; NL, naturally lactating; HIL, hormone-inducedThe pharmacokinetics by which drugs and their metabolites or vaccine-elicited immune responses appear in the breast milk of lactating women is currently conducted only in aftermarket studies. Using lactating NHP for these studies would reveal pharmacokinetics, metabolism, and host responses in the milk compartment before the drug was marketed and used clinically in lactating women. However, the resources associated with using breeding female NHP for these types of studies are considerable. A much more time-efficient and cost-effective approach that spares the fetus or infant from exposure to experimental interventions is the induction of lactation in nonpregnant NHP. Moreover, this approach spares additional breeding solely for studies of lactation. In fact, this lactation induction protocol has been used successfully in several studies to explore immunologic and virologic mechanisms of postnatal virus transmission in SIV-infected and HIV1-vaccinated monkeys, with the goal of developing strategies to reduce breast-milk transmission of HIV1.^{1,4,6,11,28,29}We previously described our protocol for inducing lactation in RM and AGM by using exogenous hormone treatment (that is, hormone-induced lactation [HIL]).^22-25,28,29 Increasing doses of depot medroxyprogesterone and estradiol were administered intramuscularly for 2 mo to mimic the hormone fluctuations of natural pregnancy and achieve mammary tissue development. After 2 mo of hormone injections, haloperidol was administered orally to raise serum prolactin levels and induce milk production. Oxytocin was given prior to milk collection to stimulate milk let-down. We also reported a comparison of the cellular and immunoglobulin content in the milk of naturally lactating (NL) and HIL RM. The lymphocyte distribution was similar between the 2 types of milk, yet the IgG content was higher in HIL milk²² but similar to that seen in less mature or early milk. Therefore, we concluded that this method was a good model for the assessment of immunologic parameters but that it may better represent the immunologic milieu of colostrum. The milk volumes obtained from the RM were quite variable (100 µL to 2 mL), and when this protocol was used in AGM, milk volumes collected were much less than expected (30 to 100 μL), often limiting the laboratory assays that could be performed. Therefore, optimizing the HIL protocol in the nonpregnant NHP would further facilitate pharmacologic and immunologic investigations in lactating NHP.The aims of our current study were 1) to determine the relationship between serum hormone levels (progesterone, medroxyprogesterone, estradiol, and prolactin) and other clinical factors to milk volume collected from nonpregnant RM and AGM that underwent HIL and 2) to compare the hormone levels achieved during HIL to those of NL AGM. We hypothesized that age, parity, and serum hormone levels are associated with milk volume in each species after HIL, and we aimed to determine the combination that predicted the highest milk volume. Determining the clinical factors and hormone levels that best predict high milk volume in the HIL protocol would increase the applicability of this technique, obviating the need to use breeding and infant animals in the assessment of drug levels and immune responses in milk.     

Estimation of Resource Availability with Special Reference to Nonhuman Primates in a Rainforest Region in the Central Western Ghats, South India

Phenological studies help to analyze resource abundance and feeding ecology of some herbivorous primate species. The rainforest between the rivers Aghnashini and Sharavathy in Karnataka, India, is one of the richest primate habitats in the country. Plant resource availability was studied in this forest using belt transect and plot methods. More than 800 trees of 71 species were considered for phenology sampling, and the tree species used by primates for consumption of leaves, flowers and fruits were selected for availability monitoring. Flush leaves were available throughout the year. The availability of mature leaves and flowers was highest in pre-summer (December–February). Unripe and ripe fruits were mostly available in summer (March–May). The availability of these resources showed significant seasonal variation. The availability and abundance of plant food resources throughout the year, presence of keystone species and broad food niches probably account for the presence of a high density of primates including 32, 47 and 151 groups of lion-tailed macaques, bonnet macaques and Hanuman langurs respectively in about 200 km² of this forest making this region a model for primate conservation.     

Preclinical Differences of Intravascular AAV9 Delivery to Neurons and Glia: A Comparative Study of Adult Mice and Nonhuman Primates

Other labs have previously reported the ability of adeno-associated virus serotype 9 (AAV9) to cross the blood-brain barrier (BBB). In this report, we carefully characterized variables that might affect AAV9''s efficiency for central nervous system (CNS) transduction in adult mice, including dose, vehicle composition, mannitol coadministration, and use of single-stranded versus self-complementary AAV. We report that AAV9 is able to transduce approximately twice as many neurons as astrocytes across the entire extent of the adult rodent CNS at doses of 1.25 × 10¹², 1 × 10¹³, and 8 × 10¹³ vg/kg. Vehicle composition or mannitol coadministration had only modest effects on CNS transduction, suggesting AAV9 crosses the BBB by an active transport mechanism. Self-complementary vectors were greater than tenfold more efficient than single-stranded vectors. When this approach was applied to juvenile nonhuman primates (NHPs) at the middle dose (9–9.5 × 10¹² vg/kg) tested in mice, a reduction in peripheral organ and brain transduction was observed compared to mice, along with a clear shift toward mostly glial transduction. Moreover, the presence of low levels of pre-existing neutralizing antibodies (NAbs) mostly occluded CNS and peripheral transduction using this delivery approach. Our results indicate that high peripheral tropism, limited neuronal transduction in NHPs, and pre-existing NAbs represent significant barriers to human translation of intravascular AAV9 delivery.