Visceral and neural larva migrans in rhesus macaques

Large ascarid larvae within granulomas were noted histologically in the mesenteric and pancreatic lymph nodes of 13 of 21 rhesus macaques (Macaca mulatta) euthanized as part of an experimental viral pathogenesis study. In addition, 7 of the 13 monkeys had cerebral granulomas, which in 4 animals contained nematode larvae similar to those within the lymph nodes. Despite the lesions, the animals did not show clinical signs associated with the parasitic infections. Characteristics of the larvae included, on cross-section, a midbody diameter of approximately 60 to 80 mum, a centrally located and slightly compressed intestine flanked on either side by large triangular excretory columns, and prominent single lateral cuticular alae. The morphology of the larvae was compatible with Baylisascaris spp. Baylisascariasis is a well-described infection of animals and humans that is caused by migrating larvae of the raccoon roundworm, Baylisascaris procyonis. A similar species, B. columnaris, is found in skunks and can cause cerebrospinal nematodiasis, but most reported cases of baylisascariasis have been due to B. procyonis. Our macaques were born free-ranging on an island in the southeastern United States where raccoons, but not skunks, were found to be common inhabitants, indicating that B. procyonis was the most likely parasite involved. These cases are similar to the low-level or covert cases of Baylisascaris infection described to occur in humans and provide further evidence of the existence of this parasite in the southeastern United States.     

Use of animal-operated folding perches by rhesus macaques (Macaca mulatta).

Providing captive or laboratory animals with the best possible living conditions has led to many ideas about how caging environments can be enhanced and the animals' lives can be enriched. This study focused primarily on 2 issues: more efficient use of existing caging and providing animals with a measure of control over their environments. We designed a new springloaded folding perching apparatus that, when modified for size, could be added to almost any caging system. Experiment 1 measured usage by animals in standard laboratory caging for rhesus macaque monkeys (Macaca mulatta). Experiment 2 measured usage by this same species in social groups in a 5-acre outdoor-indoor field setting, where several other forms of enrichment were available to the animals. Results indicated that the folding perches were used in both environments. Animals quickly learned to fold down the devices to use as a place to perch, even in the presence of permanent fixed perches. The folding perches did not significantly affect existing behavioral repertoires, but they altered how the animal used the cage. Increased animal presence near folding perches during experiment 2 suggests that these devices actually were preferred. The preference results can only partially be explained by novelty. The folding perches afforded animals a measure of control over their immediate environment without interfering in research or animal care efforts. Including at least 1 folding perch per cage satisfies both the letter and the spirit of regulations on environmental enhancement for captive primates.     

LFA-1-specific therapy prolongs allograft survival in rhesus macaques

Outcomes in transplantation have been limited by suboptimal long-term graft survival and toxicities associated with current immunosuppressive approaches. T cell costimulation blockade has shown promise as an alternative strategy to avoid the side effects of conventional immunosuppressive therapies, but targeting CD28-mediated costimulation alone has proven insufficient to prevent graft rejection in primates. Donor-specific memory T (TM) cells have been implicated in costimulation blockade-resistant transplant rejection, due to their enhanced effector function and decreased reliance on costimulatory signaling. Thus, we have tested a potential strategy to overcome TM cell-driven rejection by targeting molecules preferentially expressed on these cells, such as the adhesion molecule lymphocyte function-associated antigen 1 (LFA-1). Here, we show that short-term treatment (i.e., induction therapy) with the LFA-1-specific antibody TS-1/22 in combination with either basiliximab (an IL-2Rα-specific mAb) and sirolimus (a mammalian target of rapamycin inhibitor) or belatacept (a high-affinity variant of the CD28 costimulation-blocker CTLA4Ig) prolonged islet allograft survival in nonhuman primates relative to control treatments. Moreover, TS-1/22 masked LFA-1 on TM cells in vivo and inhibited the generation of alloproliferative and cytokine-producing effector T cells that expressed high levels of LFA-1 in vitro. These results support the use of LFA-1-specific induction therapy to neutralize costimulation blockade-resistant populations of T cells and further evaluation of LFA-1-specific therapeutics for use in transplantation.     

Toxicity of a first-generation adenoviral vector in rhesus macaques.

We constructed a first-generation adenovirus vector (AVC3FIX5) that we used to assess the rhesus macaque as a nonhuman primate model for preclinical testing of hemophilia B gene therapy vectors. Although we succeeded in our primary objective of demonstrating expression of human factor IX we encountered numerous toxic side effects that proved to be dose limiting. Following intravenous administration of AVC3FIX5 at doses of 3.4 x 10(11) vector particles/kg to 3.8 x 10(12) vector particles/kg, the animals in our study developed antibodies against human factor IX, and dose-dependent elevations of enzymes specific for liver, muscle, and lung injury. In addition, these animals showed dose-dependent prolongation of clotting times as well as acute, dose-dependent decreases in platelet counts and concomitant elevation of fibrinogen and von Willebrand factor. These abnormalities may be caused by the direct toxic effects of the adenovirus vector itself, or may result indirectly from the accompanying acute inflammatory response marked by elevations in IL-6, a key regulator of the acute inflammatory response. The rhesus macaque may be a useful animal model in which to evaluate mechanisms of adenovirus toxicities that have been encountered during clinical gene therapy trials.     

Pharmacokinetics of tenofovir in breast milk of lactating rhesus macaques

To study tenofovir transfer into milk, two lactating macaques were given a subcutaneous dose of tenofovir (30 mg/kg of body weight). Peak concentrations and area under the curve values of tenofovir in milk were approximately 3 and approximately 20% of those detected in serum, respectively.     

Antimalarial effects of human immunodeficiency virus protease inhibitors in rhesus macaques

The antimalarial activity of the human immunodeficiency virus protease inhibitors indinavir and saquinavir was evaluated in rhesus macaques for the first time. Indinavir effectively suppressed the growth of Plasmodium cynomolgi and Plasmodium knowlesi in vivo after a 7- or 3-day treatment, respectively, with clinically relevant doses, whereas saquinavir showed only weak activity against P. cynomolgi.     

Simian foamy virus infection by whole-blood transfer in rhesus macaques: potential for transfusion transmission in humans

BACKGROUND: Cross-species infection of humans with simian foamy virus (SFV) has been reported in European and North American nonhuman primate (NHP) handlers, primarily due to wound injuries involving infected animals in research centers and zoos. Additionally, African hunters have been found to be infected with SFV by exposure to body fluids, blood, or tissues of infected NHPs in the wild. The persistence of infectious virus in peripheral blood mononuclear cells (PBMNC) and the recent identification of some infected blood donors has raised safety concerns regarding potential virus transmission by blood transfusion. STUDY DESIGN AND METHODS: SFV infection by blood transfusion was evaluated by whole-blood transfer from two naturally-infected rhesus macaques (designated as D1 and D2) to retrovirus-free monkeys. Blood from D1 was transfused to two recipient monkeys R1 and R2 and from D2 to monkeys R3 and R4. Virus transmission was evaluated by immunoassays, polymerase chain reaction assays, and coculture of PBMNC for SFV isolation. RESULTS: SFV infection was seen in R1 and R2 based on development of virus-specific antibodies, identification of SFV sequences in monkey PBMNC, and isolation of infectious virus from PBMNC. Furthermore, both R1 and R2 remained SFV-positive at about 1 year after transfusion, which was the last time tested. No evidence of SFV infection was seen in R3 and R4. CONCLUSION: SFV transmission in macaques occurred by transfusion of blood from one of two infected donor animals. These results indicate the potential of SFV transfusion transmission in humans, which may depend on virus-specific or donor-related factors.     

Complications of gastric catheters implanted in rhesus macaques (Macaca mulatta).

As part of a study addressing chronic alcohol consumption and simian immunodeficiency virus, 31 rhesus macaques (Macaca mulatta) were implanted with gastric catheters used to deliver alcohol or isocaloric sucrose (control). Once implanted, the animals wore jackets and were housed in specialized cages modified with swivels and tethers. During the course of the study, 3 animals developed clinical signs indicating possible instability of the implanted gastric catheter. All 3 animals were found to have a string foreign body wrapped around the distal end of the catheter, with 2 of the catheters perforating the intestinal wall. Gastroscopy was used to screen remaining animals to determine catheter position and the presence of a foreign body attached to the end of the catheter. Results of the screening revealed that of the 28 remaining animals, 9 had malpositioned catheters; string foreign bodies were associated with 3 of the 9 malpositioned catheters. We initially hypothesized that the peristaltic motion of the stomach, combined with the attachment of string, which was probably ingested by subjects after manipulating their jackets, led to eventual catheter displacement. We later concluded that the string may have played a secondary role but was not the primary cause of catheter instability, because several malpositioned catheters had no string attached at the time of diagnosis. Subsequent modifications were instituted, including modifying the surgical technique, altering the type of gastric catheter used, and increasing environmental enrichment for animals with known tendency to manipulate their jackets.     

Site-specific metabolism of naphthalene and 1-nitronaphthalene in dissected airways of rhesus macaques

Studies in rodents have demonstrated the importance of cytochrome P450 monooxygenases in generating reactive metabolites that produce Clara cell injury. Pulmonary P450 activities in rodents are much higher than those in primates, raising the issue of relevance of rodent data to primates. Few studies on P450-catalyzed activation of cytotoxicants in subcompartments of primate lung have been reported. Accordingly, infant monkey airway subcompartments, including trachea, proximal, midlevel, distal airways, and parenchyma, were incubated with naphthalene or 1-nitronaphthalene to define metabolism at both high (500 microM) and low (50 microM) substrate concentrations. There was a relatively even distribution of metabolizing activities for naphthalene across subcompartments, but at high concentrations of 1-nitronaphthalene, lower airways (midlevel airway through parenchyma) showed higher bioactivation than upper airways. Dihydrodiol was the predominant water-soluble metabolite of naphthalene generated by all subcompartments, whereas covalently bound metabolites accounted for the greatest percentage of 1-nitronaphthalene metabolites, especially in lower airways. As anticipated, the amounts of metabolite covalently bound as a percentage of total metabolite formed increased dramatically with the 10-fold increase in substrate concentration. With both substrates, the formation of water-soluble metabolites was approximately 100 times less than observed previously in rodents. We conclude that 1) there are significant quantitative differences between rhesus and rodents in substrate bioactivation; 2) the distribution of metabolizing activities for naphthalene but not 1-nitronaphthalene is significantly different for rodents and primates; and 3) a very high percentage of the metabolites generated, particularly for 1-nitronaphthalene, is bound covalently to cellular proteins.     

Low-dose rectal inoculation of rhesus macaques by SIVsmE660 or SIVmac251 recapitulates human mucosal infection by HIV-1

We recently developed a novel strategy to identify transmitted HIV-1 genomes in acutely infected humans using single-genome amplification and a model of random virus evolution. Here, we used this approach to determine the molecular features of simian immunodeficiency virus (SIV) transmission in 18 experimentally infected Indian rhesus macaques. Animals were inoculated intrarectally (i.r.) or intravenously (i.v.) with stocks of SIVmac251 or SIVsmE660 that exhibited sequence diversity typical of early-chronic HIV-1 infection. 987 full-length SIV env sequences (median of 48 per animal) were determined from plasma virion RNA 1–5 wk after infection. i.r. inoculation was followed by productive infection by one or a few viruses (median 1; range 1–5) that diversified randomly with near starlike phylogeny and a Poisson distribution of mutations. Consensus viral sequences from ramp-up and peak viremia were identical to viruses found in the inocula or differed from them by only one or a few nucleotides, providing direct evidence that early plasma viral sequences coalesce to transmitted/founder viruses. i.v. infection was >2,000-fold more efficient than i.r. infection, and viruses transmitted by either route represented the full genetic spectra of the inocula. These findings identify key similarities in mucosal transmission and early diversification between SIV and HIV-1, and thus validate the SIV–macaque mucosal infection model for HIV-1 vaccine and microbicide research.An effective HIV-1 vaccine, microbicide, or other pre- or post-exposure prophylactic must interdict virus at or near the moment of mucosal transmission or in the early period preceding the establishment of viral latency and disseminated infection (1–4). In humans, it has been difficult to study these earliest viral host events in vivo (2, 5–13), and in tissue explant cultures or in Indian rhesus macaques the HIV-1 or simian immunodeficiency virus (SIV) inocula have typically been high to achieve uniform infection of controls or to visualize infection events in situ (14–20), thus prompting concerns about the physiological relevance of the model systems (21–24). Further complicating the analysis of early infection events in vivo is the viral “eclipse” period during which virus replicates in mucosal and locoregional lymphoreticular tissues but is not yet detectable in the circulating plasma (25). In SIV-infected macaques, the eclipse period is generally ∼4–7 d in duration, and in HIV-1–infected humans, it is ∼7–21 d (5, 18, 25–27).Previously, we observed that in the early stages of HIV-1 infection preceding antibody seroconversion (eclipse phase and Fiebig stages I and II [25]), virus diversification follows a pattern of random evolution with an almost starlike phylogeny and a Poisson distribution of nucleotide substitutions (5). We thus hypothesized that the genetic identity of transmitted or early founder viruses could be inferred unambiguously by phylogenetic analysis of discrete low-diversity viral lineages that emanate from them. This hypothesis was supported by an analysis of 3,449 full-length env genes from 102 human subjects with acute HIV-1 subtype B infection, where we found that (a) acute viral sequences sampled before the development of measurable adaptive immune responses conformed to a pattern of random virus evolution; (b) viral sequence diversity resulted in model estimates of time to a most recent common ancestor (MRCA) that was consistent with clinical histories and Fiebig stage classifications; and (c) in most subjects (78 of 102) there was evidence of productive infection by only a single virus, whereas in 24 other subjects infection resulted from transmission of at least 2 to 5 viruses, each recognizable as a discrete virus lineage (5). These findings have since been corroborated in seven additional patient cohorts infected by HIV-1 subtypes A, B, C, or D (28–35). A key innovation common to these studies was the use of singe-genome amplification (SGA) of plasma viral RNA, followed by direct amplicon sequencing to characterize the virus quasispecies (5, 35–39). This method provides proportional representation of plasma viral RNA (vRNA) and precludes Taq polymerase-induced template switching (recombination), Taq polymerase-associated nucleotide substitutions in finished sequences, template resampling, and cloning bias (5, 35, 37, 38, 40).In this study, we sought to directly test our strategy for identifying transmitted/founder viruses in the Indian rhesus macaque SIV infection model where we could define essential experimental parameters, including the route of SIV infection, genetic composition of the inoculum, and the duration between virus inoculation and sampling of plasma vRNA. The primary study objectives were twofold: first, to determine if, as our hypothesis and model predict, plasma SIV sequences sampled at or near peak viremia coalesce to sequences of viruses responsible for transmission and productive clinical infection weeks earlier; and second, to determine how closely a low-dose SIV rectal transmission model in Indian rhesus macaques recapitulates features of human infection by HIV-1, including the extent of the mucosal barrier to virus transmission, the number of transmitted/founder viruses leading to productive infection, and the molecular patterns of early virus diversification.     

Prediction and validation of hematopoietic stem and progenitor cell off-target editing in transplanted rhesus macaques

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A pharmacological analysis of the discriminative stimulus properties of d-amphetamine in rhesus monkeys

Rhesus monkeys (n = 4) were trained to discriminate d-amphetamine (AMPH; 0.67 or 1.33 mumol/kg i.v.) from saline in a two lever, food-reinforced drug discrimination paradigm. After acquisition of the discrimination (average, 127 sessions), the monkeys were tested with a series of compounds selected to characterize the neuronal mechanism(s) of the discrimination. AMPH (0.08-2.6 mumol/kg), cocaine (0.06-1.0 mumol/kg; n = 4), the dopamine (DA) uptake inhibitor bupropion (0.25-2.0 mumol/kg; n = 2) and the norepinephrine (NE) uptake inhibitor nisoxetine (1.0-16 mumol/kg; n = 4) produced dose-related increases in the percentage of responses that occurred on the AMPH-appropriate lever during test sessions in all monkeys tested. For all other drugs tested, individual differences in effects were noted. Compounds with primarily D2 DA receptor activity, including apomorphine (0.06-1.0 mumol/kg; n = 4), piribedil (0.25-8.0 mumol/kg; n = 4), bromocriptine (0.12-1.0 mumol/kg; n = 4) and propylbutyldopamine (0.25-4.0 mumol/kg; n = 3) occasioned AMPH-appropriate responding in one or two monkeys. The D1 agonist SKF 38393 (0.5-64 mumol/kg; n = 4) and pentobarbital (4.0-32 mumol/kg; n = 4) substituted for AMPH in only one monkey. In tests for antagonism, the D1 antagonist SCH 23390 (0.015-0.03 mumol/kg; n = 2) blocked completely the discriminative stimulus effects of AMPH in a dose-related manner.(ABSTRACT TRUNCATED AT 250 WORDS)     

Preclinical evaluation of adult stem cell engraftment and toxicity in the CNS of rhesus macaques.

Congenital neurodegenerative diseases exhibit progressive postnatal neurologic impairment leading to premature death and are intractable to systemic therapies such as bone marrow transplantation. We injected bone marrow-derived mesenchymal stem cells (MSCs) into the CNS of young adult rhesus macaques to evaluate their safety and feasibility as vectors for direct intervention of neurologic disorders. Levels of engrafted male, donor MSCs were quantified in the CNS of female transplant recipients by real-time PCR using an SRY gene-specific probe. Analysis of coronal brain slices encompassing one-third of the total brain volume revealed engraftment levels ranging from 0.026 x 10(-3) to 0.163 x 10(-3)% of the total DNA content of brain tissue. Fine-mapping revealed male DNA distributed within specific anatomic structures along the neuraxis where label-retaining MSCs were visualized in histological sections by immunohistochemistry. Double labeling of sections confirmed that engrafted donor cells lacked expression of the macrophage marker CD68, the astrocytes marker GFAP, and neuronal markers NeuN and MAP2. MSC engraftment had no adverse effects on animal health, behavior, postural and locomotor patterns, or upper limb motor performance evaluated over a 6-month period posttransplantation. Therefore, MSC-based therapies represent a safe alternative for clinical intervention of CNS disorders.     

Heterologous prime-boost strategy to immunize very young infants against measles: pre-clinical studies in rhesus macaques

Infants in developing countries are at high risk of developing severe clinical measles if they become infected during the "window of vulnerability" (age 4-9 months), when declining maternal antibodies do not protect against wild virus, yet impede successful immunization by attenuated measles vaccine. We developed two Sindbis replicon-based DNA vaccines expressing measles virus hemagglutinin and fusion protein with the goal of priming young infants to respond safely and effectively to subsequent boosting with attenuated measles vaccine. Intradermal prime with DNA vaccines by needle-free injection followed by aerosol or parenteral boost with licensed measles vaccine was well tolerated by juvenile and young infant rhesus macaques, and protected against clinical measles and viremia on wild-type virus challenge. A proteosome-measles vaccine administered alone (three doses) or as a boost following DNA vaccine priming was also safe and protective. These promising results pave the way for clinical trials to assess this prime-boost strategy.     

Transendocardial delivery of AAV6 results in highly efficient and global cardiac gene transfer in rhesus macaques

Heart disease is the leading cause of morbidity and mortality, and cardiac gene transfer has potential as a novel therapeutic approach. We previously demonstrated safe and efficient gene transfer to the canine heart using a percutaneous transendocardial injection procedure to deliver self-complementary (sc) adeno-associated virus 6 (AAV6) vector. In the present study, we proceed with our vertical translation study to evaluate cardiac gene transfer in nonhuman primates (NHPs). We screened approximately 30 adult male rhesus macaques for the presence of neutralizing antibodies against AAV6, AAV8, and AAV9, and then selected seven monkeys whose antibody titers against these three serotypes were lower than 1/5. The animals were then randomized to receive either scAAV6 (n=3), scAAV8 (n=1), or scAAV9 (n=3) vector expressing the enhanced green fluorescent protein (EGFP) reporter gene at a dose of 5.4×10(12) genome copies/kg, which was administered according to a modified version of our previously developed transendocardial injection procedure. One animal treated with scAAV6 died secondary to esophageal intubation. The remaining animals were euthanized 7 days after gene transfer, at which time tissue was collected for analysis of EGFP expression, histopathology, and biodistribution of the vector genome. We found that (i) transendocardial delivery of AAV is safe in the NHP, (ii) AAV6 and AAV8 provide efficient cardiac gene transfer at similar levels and are superior to AAV9, and (iii) AAV6 is more cardiac-specific than AAV8 and AAV9. The results of this NHP study may help guide the development AAV vectors for the treatment of cardiovascular disease in humans.     

Intranasal vaccination with AAV5 and 9 vectors against human papillomavirus type 16 in rhesus macaques

Cervical cancer is the second most common cancer in women worldwide. Persistent high-risk human papillomavirus (HPV) infection has been identified as the causative event for the development of this type of cancer. Recombinant adeno-associated viruses (rAAVs) are currently being developed and evaluated as vaccine vector. In previous work, we demonstrated that rAAVs administered intranasally in mice induced high titers and long-lasting neutralizing antibodies against HPV type 16 (HPV16). To extend this approach to a more human-related species, we immunized rhesus macaques (Macaca mulatta) with AAVs expressing an HPV16 L1 protein using rAAV5 and 9 vectors in an intranasal prophylactic setting. An rAAV5-L1 vector followed by a boost with rAAV9-L1 induced higher titers of L1-specific serum antibodies than a single rAAV5-L1 immunization. L1-specific antibodies elicited by AAV9 vector neutralized HPV16 pseudovirions and persisted for at least 7 months post immunization. Interestingly, nasal application of rAAV9 was immunogenic even in the presence of high AAV9 antibody titers, allowing reimmunization with the same serotype without prevention of the transgene expression. Two of six animals did not respond to AAV-mediated intranasal vaccination, although they were not tolerant, as both developed antibodies after intramuscular vaccination with HPV16 virus-like particles. These data clearly show the efficacy of an intranasal immunization using rAAV9-L1 vectors without the need of an adjuvant. We conclude from our results that rAAV9 vector is a promising candidate for a noninvasive nasal vaccination strategy.     

Cocaine-reinforced responding in rhesus monkeys: pharmacological attenuation of the hypothalamic-pituitary-adrenal axis response.

Intravenously self-administered cocaine produces a dose-dependent release of adrenocorticotropic hormone (ACTH) and cortisol in male rhesus monkeys. This study investigated whether the acute disruption of cortisol and/or ACTH release had any effect on ongoing cocaine-maintained responding. Four hypothalamic-pituitary-adrenal (HPA) axis inhibitors were examined: etomidate and ketoconazole, both of which are cortisol synthesis inhibitors; astressin, a peptidic corticotropin-releasing factor (CRF) antagonist that binds CRF(1) receptors predominantly in the pituitary gland; and dexamethasone, a highly selective glucocorticoid receptor agonist whose long-lasting effects reduce or abolish the endogenous release of ACTH and cortisol. The reinforcing effects of a range of cocaine doses, with or without pretreatment with an HPA inhibitor, were evaluated using a fixed ratio 30 time-out 10-min schedule of reinforcement in six male monkeys. Blood was sampled before, during, and after self-administration sessions. Self-administration of cocaine increased plasma cortisol and ACTH. Pretreatment with etomidate and ketoconazole dose-dependently inhibited the cocaine-induced rise in cortisol and, at the highest doses, produced a compensatory increase in ACTH release. Astressin and dexamethasone attenuated or abolished cocaine-induced cortisol and ACTH release. Despite the efficacy exhibited by these pretreatments and the variety of mechanisms by which they inhibited the HPA axis, there was no evidence for any change in cocaine-reinforced behavior (response rate or infusion number), an indication that acute changes in the ACTH or cortisol response to cocaine do not play a direct role in modulating cocaine-seeking behavior under these behavioral circumstances.