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Nonbronchoscopic bronchoalveolar lavage (NB-BAL) is a minimally invasive diagnostic and research tool used to sample the cells of lower airways and alveoli without using a bronchoscope. Our study compared NB-BAL and bronchoscopic bronchoalveolar lavage (B-BAL) in terms of costs, cell yields, and the number of post-procedural complications in macaques. We also analyzed procedure times, BAL fluid volume yields, and vital signs in a subset of animals that underwent NB-BAL. Compared with the B-BAL technique, NB-BAL was less expensive to perform, with fewer complications, fewer animals requiring temporary or permanent cessation of BALs, and higher cell yields per mL of recovered saline. The average procedure time for NB-BAL was 6.8 ± 1.6 min, and the average NB-BAL lavage volume yield was 76 ± 9%. We found no significant differences in respiration rate before, during, or after NB-BAL but did find significant differences in heart rate and oxygen saturation (SpO2). This study demonstrates that NB-BAL is a simple, cost-effective, and safe alternative to B-BAL that results in higher cell yields per mL, improved animal welfare, and fewer missed time points, and thus constitutes a refinement over the B-BAL in macaques.

Macaques are physiologically relevant animal models of human immunology and infectious disease that have provided key insights and advanced clinical treatment in transplantation, vaccinology, and HIV/AIDS. However, the small size of macaques is a stumbling block for studies requiring large numbers of cells, such as hematopoietic stem cells (HSCs) for transplantation, antigen-specific lymphocytes for in-depth immunological analysis, and latently-infected CD4+ T-cells for HIV cure studies. Here, we provide a detailed protocol for collection of large numbers of HSCs and T-cells from cynomolgus macaques as small as 3 kilograms using the Terumo Spectra Optia apheresis system, yielding an average of 5.0 x 10(9) total nucleated cells from mobilized animals and 1.2 x 10(9) total nucleated cells from non-mobilized animals per procedure. This report provides sufficient detail to adapt this apheresis technique at other institutions, which will facilitate more efficient and detailed analysis of HSCs and their progeny blood cells.

Ultrasound (US) generated by catheters used clinically for US-facilitated thrombolysis can release shear-dependent vasodilators from endothelial and red blood cells. We hypothesized that catheter-based US in the pulmonary artery (PA) decreases downstream vascular resistance and increases pulmonary blood flow. In rhesus macaques, a U.S. Food and Drug Administration-approved multi-element US catheter was placed in a pulmonary artery. Comprehensive echocardiography was performed (i) at baseline, (ii) during hypoxemia (12% FIOsub2/sub) to increase pulmonary vascular resistance (PVR) and (c) 15 min after initiating US during hypoxemia. Reduced FIOsub2/subproduced intended reductions in oxygen saturation (69 ± 3%) and PaOsub2/sub(34 ± 5 mm Hg), yet on echocardiography, hypoxemia did not create the intended model, with only modest hypoxia-related increases in PA systolic pressure (24 ± 4 to 28 ± 4 mm Hg, p = 0.05) and no significant change in PVR or multiparametric right ventricular (RV) function. Although US did not further change total PVR, onsup99m/supTc-macroalbumin aggregate single-photon-emission computed tomography imaging, lung perfusion was significantly higher in the lung ipsilateral to the US catheter versus the contralateral control lung (133 ± 48 cpm vs. 103 ± 43 × 10sup3/supcpm, p = 0.01). We conclude that PA catheter-based US increases regional lung perfusion, most likely from vasodilators that are conducted downstream.

Ultrasound (US) is known to stimulate endogenous shear-dependent pathways, and can lower microvascular resistance through mediators that are conducted downstream from US exposure. We hypothesized that endovascular US already in use for thrombolysis in humans, can improve tissue perfusion in the setting of acute limb ischemia through downstream-conducted effects. Models of severe peripheral arterial disease were developed in mice and in rhesus macaques. An endovascular US catheter (2.3 MHz, 0.5 to 1.1 MPa) was used to expose the limb adductor in mice for 10 minutes; or the femoral artery distal to stenosis in macaques for 15 minutes. Quantitative contrast-enhanced ultrasound perfusion imaging was performed to assess flow augmentation in the adductor muscle of mice and the calf muscle of macaques. Microvascular blood flow in the ischemic limb relative to the contralateral control limb was reduced to 22±8% in mice, and 36±20% in macaques. US produced an immediate 2.3-fold and 3-fold increase (p

National Institutes of Health (NIH) from the National Institute of Allergy and Infectious Diseases (NIAID) [R21 All 28970-01A1]; National Institutes of Health (NIH) from the NIH Office, and Martin Delaney BELIEVE collaborator NIAID award [P51 OD011092, UM1A1126617]; National Institute on Drug Abuse, National Institute of Mental Health, and National Institute of Neurological Disorders and Stroke

Allogeneic hematopoietic stem cell transplantation (HSCT) and xenotransplantation are accompanied by viral reactivations and virus-associated complications resulting from immune deficiency. Here, in a Mauritian cynomolgus macaque model of fully MHC-matched allogeneic HSCT, we report reactivations of cynomolgus polyomavirus, lymphocryptovirus, and cytomegalovirus, macaque viruses analogous to HSCT-associated human counterparts BK virus, Epstein-Barr virus, and human Cytomegalovirus. Viral replication in recipient macaques resulted in characteristic disease manifestations observed in HSCT patients, such as polyomavirus-associated hemorrhagic cystitis and tubulointerstitial nephritis or lymphocryptovirus-associated post-transplant lymphoproliferative disorder. However, in most cases, the reconstituted immune system, alone or in combination with short-term pharmacological intervention, exerted control over viral replication, suggesting engraftment of functional donor-derived immunity. Indeed, the donor-derived reconstituted immune systems of two long-term engrafted HSCT recipient macaques responded to live attenuated yellow fever 17D vaccine (YFV 17D) indistinguishably from untransplanted controls, mounting 17D-targeted neutralizing antibody responses and clearing YFV 17D within 14 days. Together, these data demonstrate that this macaque model of allogeneic HSCT recapitulates clinical situations of opportunistic viral infections in transplant patients, and provides a pre-clinical model to test novel prophylactic and therapeutic modalities.

Allogeneic hematopoietic stem cell transplantation (HSCT) is a critically important therapy for hematological malignancies, inborn errors of metabolism, and immunodeficiency disorders, yet complications such as graft-vs.-host disease (GvHD) limit survival. Development of anti-GvHD therapies that do not adversely affect susceptibility to infection or graft-vs.-tumor immunity are hampered by the lack of a physiologically relevant, preclinical model of allogeneic HSCT. Here we show a spectrum of diverse clinical HSCT outcomes including primary and secondary graft failure, lethal GvHD, and stable, disease-free full donor engraftment using reduced intensity conditioning and mobilized peripheral blood HSCT in unrelated, fully MHC-matched Mauritian-origin cynomolgus macaques. Anti-GvHD prophylaxis of tacrolimus, post-transplant cyclophosphamide, and CD28 blockade induces multi-lineage, full donor chimerism and recipient-specific tolerance while maintaining pathogen-specific immunity. These results establish a new preclinical allogeneic HSCT model for evaluation of GvHD prophylaxis and next-generation HSCT-mediated therapies for solid organ tolerance, cure of non-malignant hematological disease, and HIV reservoir clearance., Rhesus macaques are not ideal for studying response to allogeneic hematopoietic stem cell transplant (allo-HSCT) owing to complex MHC genetics that prevent full MHC-matching. Here the authors show that inbred Mauritian-origin cynomolgus macaques are a superior preclinical model of allogeneic stem cell transplantation that mimics diverse clinical outcomes of human allo-HSCT.

The strict tropism of many pathogens for man hampers the development of animal models that recapitulate important microbe–host interactions. We developed a rhesus macaque model for studying Neisseria –host interactions using Neisseria species indigenous to the animal. We report that Neisseria are common inhabitants of the rhesus macaque. Neisseria isolated from the rhesus macaque recolonize animals after laboratory passage, persist in the animals for at least 72 d, and are transmitted between animals. Neisseria are naturally competent and acquire genetic markers from each other in vivo, in the absence of selection, within 44 d after colonization. Neisseria macacae encodes orthologs of known or presumed virulence factors of human-adapted Neisseria , as well as current or candidate vaccine antigens. We conclude that the rhesus macaque model will allow studies of the molecular mechanisms of Neisseria colonization, transmission, persistence, and horizontal gene transfer. The model can potentially be developed further for preclinical testing of vaccine candidates.

Ischemia-reperfusion brain injury can be iatrogenically induced secondary to life-saving procedures. Prophylactic treatment of these patients offers a promising prevention for lifelong complications. We postulate that a cytosine-guanine (CpG) oligodeoxynucleotide (ODN) can provide robust antecedent protection against cerebral ischemic injury with minimal release of pro-inflammatory cytokines, making it an ideal candidate for further clinical development. Mouse and nonhuman primate (NHP) models of cerebral ischemic injury were used to test whether an A-type CpG ODN, which induces minimal systemic inflammatory cytokine responses, can provide prophylactic protection. Extent of injury in the mouse was measured by histological staining of live tissue. In the NHP, injury was assessed 2 and 7 days post-occlusion from T2-weighted magnetic resonance images and neurological and motor deficits were cataloged daily. Plasma cytokine levels were measured using species-specific Luminex assays. Prophylactic administration of an A-type CpG ODN provided robust protection against cerebral ischemic injury in the mouse with minimal systemic inflammation. Rhesus macaques treated with D192935, a mixture of human optimized A-type CpG ODNs, had smaller infarcts and demonstrated significantly less neurological and motor deficits following ischemic injury. Our findings demonstrate the translational potential of D192935 as a prophylactic treatment for patients at risk of cerebral ischemic injury.

To date, a therapy for Huntington's disease (HD), a genetic, neurodegenerative disorder, remains elusive. HD is characterized by cell loss in the basal ganglia, with particular damage to the putamen, an area of the brain responsible for initiating and refining motor movements. Consequently, patients exhibit a hyperkinetic movement disorder. RNA interference (RNAi) offers therapeutic potential for this disorder by reducing the expression of HTT, the disease-causing gene. We have previously demonstrated that partial suppression of both wild-type and mutant HTT in the striatum prevents behavioral and neuropathological abnormalities in rodent models of HD. However, given the role of HTT in various cellular processes, it remains unknown whether a partial suppression of both alleles will be safe in mammals whose neurophysiology, basal ganglia anatomy, and behavioral repertoire more closely resembles that of a human. Here, we investigate whether a partial reduction of HTT in the normal non-human primate putamen is safe. We demonstrate that a 45% reduction of rhesus HTT expression in the mid- and caudal putamen does not induce motor deficits, neuronal degeneration, astrogliosis, or an immune response. Together, these data suggest that partial suppression of wild-type HTT expression is well tolerated in the primate putamen and further supports RNAi as a therapy for HD.

Cerebral ischemic injury is a significant portion of the burden of disease in developed countries; rates of mortality are high and the costs associated with morbidity are enormous. Recent therapeutic approaches have aimed at mitigating the extent of damage and/or promoting repair once injury has occurred. Often, patients at high risk of ischemic injury can be identified in advance and targeted for antecedent neuroprotective therapy. Agents that stimulate the innate pattern recognition receptor, Toll-like receptor 9, have been shown to induce tolerance (precondition) to ischemic brain injury in a mouse model of stroke. Here, we demonstrate for the first time that pharmacological preconditioning against cerebrovascular ischemic injury is also possible in a nonhuman primate model of stroke in the rhesus macaque. The model of stroke used is a minimally invasive transient vascular occlusion, resulting in brain damage that is primarily localized to the cortex and as such, represents a model with substantial clinical relevance. Finally, K-type (also referred to as B-type) cytosine-guanine-rich DNA oligonucleotides, the class of agents employed in this study, are currently in use in human clinical trials, underscoring the feasibility of this treatment in patients at risk of cerebral ischemia.

The ovarian surface epithelium (OSE) has a prominent role in ovarian cancer in women, but no studies have been conducted to evaluate its role in normal ovarian function. Data from other species suggest the OSE is needed for ovulation. We have tested whether the OSE is needed for follicle rupture, a necessary step in ovulation, using the nonhuman primate, rhesus macaque. The OSE was removed in two different short-term protocols spanning a single periovulatory interval—one protocol used a cytology brush to remove the OSE only from the follicle apex, and one used mild detergent to remove the entire OSE—and in one long-term protocol spanning 6 wk (two periovulatory intervals) that removed the entire OSE with detergent. Serum levels of estrogen and progesterone (E and P) were monitored, and sectioned ovaries were examined for evidence of successful OSE removal and follicle rupture. In the short-term protocols, removal of the OSE over the follicle apex did not prevent follicle rupture (n = 4 ovaries), but removal of the entire OSE using detergent did in four of six cases. In the long-term protocol, when ovaries were collected after the second periovulatory interval, all the ovaries (n = 5) showed evidence of follicle rupture. In all the protocols, E and P production appeared unaffected. Detergent penetrated up to 40 μm into the ovary. This may have transiently disrupted the stroma and caused follicle rupture failure. We conclude that the primate OSE is not essential for ovulation and perhaps can be removed without lasting consequence.

Primate models are essential tools for translational research in stroke but are reportedly inconsistent in their ability to produce cortical infarcts of reproducible size. Here, we report a new stroke model using a transorbital, reversible, two-vessel occlusion approach in male rhesus macaques that produces consistent and reproducible cortical infarcts. The right middle cerebral artery (distal to the orbitofrontal branch) and both anterior cerebral arteries were occluded with vascular clips. Bilateral occlusion of the anterior cerebral artery was critical for reducing collateral flow to the ipsilateral cortex. Reversible ischemia was induced for 45, 60, or 90 mins ( n = 2/timepoint) and infarct volume and neurologic outcome were evaluated. The infarcts were located predominantly in the cortex and increased in size with extended duration of ischemia determined by T2-weighted magnetic resonance imaging. Infarct volume measured by 2,3,5-triphenyl tetrazolium chloride and cresyl violet staining corroborated magnetic resonance imaging results. Neurologic deficit scores worsened gradually with longer occlusion times. A subset of animals ( n = 5) underwent 60 mins of ischemia resulting in consistent infarct volumes primarily located to the cortex that correlated well with neurologic deficit scores. This approach offers promise for evaluating therapeutic interventions in stroke.

Most biomedical facilities that use rhesus macaques (Macaca mulatta) limit the amount of blood that may be collected for experimental purposes. These limits typically are expressed as a percentage of blood volume (BV), estimated by using a fixed ratio of blood (mL) per body weight (kg). BV estimation ratios vary widely among facilities and typically do not factor in variables known to influence BV in humans: sex, age, and body condition. We used indicator dilution methodology to determine the BV of 20 adult rhesus macaques (10 male, 10 female) that varied widely in body condition. We measured body composition by using dual-energy X-ray absorptiometry, weight, crown-to-rump length, and body condition score. Two indicators, FITC-labeled hydroxyethyl starch (FITC-HES) and radioiodinated rhesus serum albumin ((125)I-RhSA), were injected simultaneously, followed by serial blood collection. Plasma volume at time 0 was determined by linear regression. BV was calculated from the plasma volume and Hct. We found that BV calculated by using FITC-HES was consistently lower than BV calculated by using (125)I-RhSA. Sex and age did not significantly affect BV. Percentage body fat was significantly associated with BV. Subjects categorized as having 'optimal' body condition score had 18% body fat and 62.1 mL/kg BV (by FITC-HES; 74.5 mL/kg by (125)I-RhSA). Each 1% increase in body fat corresponded to approximately 1 mL/kg decrease in BV. Body condition score correlated with the body fat percentage (R(2) = 0.7469). We provide an equation for calculating BV from weight and body condition score.

We tested the hypothesis that fetal pulmonary arterial circulation reacts to changes in fetal oxygenation status at near-term gestation. A total of 20 rhesus macaques underwent fetal Doppler ultrasonography at near-term gestation. Right pulmonary artery (RPA), umbilical artery (UA), ductus arteriosus (DA), and ductus venosus (DV) blood velocity waveforms were obtained, and pulsatility index (PI) values were calculated. Fetal right and left ventricular cardiac outputs were determined. Ultrasonographic data were collected during 3 maternal oxygenation states: room air (baseline), hyperoxemia, and hypoxemia. Fetal RPA PI values increased (P < .05) during maternal hypoxemia and decreased (P < .05) during maternal hyperoxemia, compared with baseline. Maternal hyperoxemia increased (P < .05) DA PI values from baseline. Fetal cardiac outputs, UA, and DV PI values were not affected. Our results demonstrate that at near-term gestation, fetal pulmonary arterial circulation is a dynamic vascular bed that reflects acute and short-term changes in fetal oxygenation.

background: The fifth leading cause of cancer deaths among women is ovarian cancer (OC), which originates primarily in the ovarian surface epithelium (OSE) that surrounds the ovary. Permanent removal of the OSE could provide a novel strategy to substantially reduce OC risk, while retaining the benefits of ovarian function, including gameto- and steroidogenesis. It must be determined whether ovarian surface epitheliectomy (OSEx) carries deleterious side effects, including loss of menstrual cyclicity, infertility or scarring (e.g. adhesions), prior to any clinical application of this strategy. To achieve this, we selected the non-human primate, rhesus macaque, for long-term (12 month) studies on the effects of OSEx. methods: Rhesus macaque females underwent OSEx by detergent treatment and were then monitored for menstrual cyclicity (menstruation, steroidogenesis and follicle development) and adverse side effects (tissue scarring or adhesions). Ovaries were collected at 6 or 12 months and examined for evidence of tissue damage, follicle rupture and regression of the corpus luteum. The ovarian surface was examined immunohistologically for signs of epithelial replacement, using markers for OSE and fimbrial epithelium (FE), a possible alternative source of pelvic tumors diagnosed as OC. results: After OSEx, menstrual cycle length, estrogen and progesterone production, follicle rupture and luteal regression appeared normal. No evidence of adhesions was seen. At 6 and 12 months post-OSEx, the ovarian surface was sparsely populated by cells expressing OSE and FE markers. Proliferative activity in this population was notably low. conclusions: OSEx may provide a novel method to reduce the risk of OC, without sacrificing ovarian function, although the effects on fertility remain to be tested. The absence of epithelial replacement via enhanced proliferation suggests OSEx does not increase malignant potential. Complete and permanent OSEx may be feasible.

Objectives: We hypothesized that fetal ventricular diastolic filling characteristics are related to fetal heart rate (FHR) and pulmonary vascular impedance. Methods: Rhesus monkeys underwent Doppler ultrasonography at near-term gestation. Tricuspid (TV) and mitral valve (MV) blood velocity waveforms were used to calculate maximum velocity (V-max) and velocity time integral (VTI) E/A ratios (early filling/ventricular filling during atrial contraction) at baseline and during maternal hyperoxygenation. Right pulmonary artery (RPA) pulsatility indices (PIs) and FHR were measured. Results: Hyperoxgenation significantly decreased FHR and RPA PI. The TV V-max and the MV VTI E/A ratios increased significantly and correlated with a decrease in FHR but not with a decrease in RPA PI. The TV and MV A-wave V-max decreased during hyperoxygenation and their decrease correlated significantly with a drop in FHR. Conclusions: Fetal ventricular diastolic filling characteristics are related to FHR but not to pulmonary vascular impedance.

Social group housing of rhesus macaques at biomedical facilities is advocated to improve the psychologic wellbeing of these intelligent and social animals. An unintended outcome of social housing in this species is increased intraspecific aggression resulting in cases of severe multiple trauma and posttraumatic shock. The metabolic correlates of oxygen debt are likely important quantifiers of the severity of posttraumatic shock and may serve as useful guides in the treatment of these cases. The purpose of this retrospective study was to evaluate venous blood lactate, base excess, bicarbonate, and pH as predictors of mortality. These 4 variables were assessed in 84 monkeys with severe traumatic injury and shock. Data were available from blood samples collected prior to resuscitation therapy and the day after resuscitation therapy. The pre- and postresuscitation therapy levels of the variables then were tested for association with 6-d survival. When measured prior to resuscitation therapy, all variables were strongly correlated with each other and had a statistically significant association with survival. No single variable had both strong specificity and high sensitivity when measured prior to resuscitation therapy. Survival analysis showed that as the number of categorical indicators of acidosis increased, 6-d survival decreased. Analysis of the 4 variables after resuscitation therapy indicated that lactate was the only variable significantly associated with survival in our study.

Non-human primates (NHPs) are an invaluable resource for the study of genetic regulation of disease mechanisms. The main disadvantage of using NHPs as a preclinical model of human disease is the difficulty of manipulating the monkey genome using conventional gene modifying strategies. Lentiviruses offer the possibility of circumventing this difficulty because they can infect and transduce either dividing or nondividing cells, without producing an immune response. In addition, lentiviruses can permanently integrate into the genome of host cells, and are able to maintain long-term expression. In this article we describe the lentiviral vectors that we use to both express transgenes and suppress expression of endogenous genes via RNA interference (RNAi) in NHPs. We also discuss the safety features of currently available vectors that are especially important when lentiviral vectors are used in a species as closely related to humans as NHPs. Finally, we describe in detail the lentiviral vector production protocol we use and provide examples of how the vector can be employed to target peripheral tissues and the brain.

A mid-abdominal mass was discovered during routine physical examination of a rhesus macaque (Macaca mulatta). Further diagnostics and exploratory laparotomy were performed, revealing a fluid-filled cyst attached to the caudal free margin of the greater omentum. Formation and pulsatile movement of white-colored circumferential bands within the wall of the cyst were observed during surgery. The cyst was removed and later was dissected. The discovery of a single invaginated scolex identified the cyst as a cysticercus. The location and characteristics of the cysticercus were consistent with the larval form of Taenia hydatigena.