Kate C. Baker

Allo-reactivity of mesenchymal stem cells in rhesus macaques is dose and haplotype dependent and limits durable cell engraftment in vivo.

The emerging paradigm that MSCs are immune privileged has fostered the use of “off-the-shelf” allogeneic MSC-based therapies in human clinical trials. However, this approach ignores studies in experimental animals wherein transplantation of MSCs across MHC boundaries elicits measurable allo-immune responses. To determine if MSCs are hypo-immunogeneic, we characterized the immune response in rhesus macaques following intracranial administration of allogeneic vs. autologous MSCs. This analysis revealed unambiguous evidence of productive allo-recognition based on expansion of NK, B and T cell subsets in peripheral blood and detection of allo-specific antibodies in animals administered allogeneic but not autologous MSCs. Moreover, the degree of MHC class I and II mismatch between the MSC donor and recipient significantly influenced the magnitude and nature of the allo-immune response. Consistent with these findings, real-time PCR analysis of brain tissue from female recipients administered varying doses of male, allogeneic MSCs revealed a significant inverse correlation between MSC engraftment levels and cell dose. Changes in post-transplant neutrophil and lymphocyte counts also correlated with dose and were predictive of overall MSC engraftment levels. However, secondary antigen challenge failed to elicit a measurable immune response in allogeneic recipients. Finally, extensive behavior testing of animals revealed no main effect of cell dose on motor skills, social development, or temperament. Collectively, these data indicate that allogeneic MSCs are weakly immunogenic when transplanted across MHC boundaries in rhesus macaques and this negatively impacts durable engraftment levels. Therefore the use of unrelated donor MSCs should be carefully evaluated in human patients.

Age-and Dose-Related Effects on MSC Engraftment Levels and Anatomical Distribution in the Central Nervous Systems of Nonhuman Primates : Identification of Novel MSC Subpopulations That Respond to Guidance Cues in Brain

Mesenchymal stem cells (MSCs) have demonstrated efficacy as therapeutic vectors in rodent models of neurological diseases, but few studies have evaluated their safety and efficacy in a relevant large animal model. Previously, we reported that MSCs transplanted to the central nervous systems (CNS) of adult rhesus macaques engrafted at low levels without adversely affecting animal health, behavior, or motor function. Herein, we injected MSCs intracranially into 10 healthy infant macaques and quantified their engraftment levels and mapped their anatomical distribution in brain by real‐time polymerase chain reaction using an sry gene‐specific probe. These analyses revealed that MSC engraftment levels in brain were on average 18‐fold higher with a maximal observed difference of 180‐fold in neonates as compared with that reported previously for young adult macaques. Moreover, engraftment levels were 30‐fold higher after injection of a low versus high cell dose and engrafted MSCs were nonrandomly distributed throughout the infant brain and localized to specific anatomical regions. Identification of unique subpopulations of macaque and human MSCs that express receptor proteins known to regulate tangential migration of interneurons may explain their migration patterns in brain. Extensive monitoring of infant transplant recipients using a battery of age appropriate tests found no evidence of any long‐term adverse effects on the health or social, behavioral, cognitive, or motor abilities of animals up to 6 months post‐transplant. Therefore, direct intracranial injection represents a safe means to deliver therapeutic levels of MSCs to the CNS. Moreover, expressed guidance receptors on MSC subpopulations may regulate migration of cells in the host brain.

Social buffering in adult male rhesus macaques (Macaca mulatta): Effects of stressful events in single vs. pair housing

Background  The purpose of this study was to test whether long‐term pair housing of male rhesus macaques ameliorated negative responses to stressful events that can occur in the course of routine husbandry or research procedures.

Physiological and behavioral effects of social introduction on adult male rhesus macaques.

Pair housing of laboratory macaques is widely considered to lead to positive changes in well‐being, yet the process of introduction is viewed as potentially stressful and risk‐prone. Behavioral and physiological data were collected on eight adult male rhesus macaques before, during, and after the process of introduction, in order to measure the initial stress of introduction as well as long‐term changes in well‐being. Socially experienced subjects, all implanted with biotelemetry devices, were studied in five successive phases: baseline (singly housed), 1 day each of protected contact and full contact introduction, post‐introduction (1–3 weeks after introduction), and settled pairs (≥20 weeks after introduction). One hundred and seventy‐six hours of behavioral data and 672 hr of heart rate data were analyzed. Fecal cortisol was also measured for the baseline, post‐introduction, and settled pair phases. All introductions were successful and subjects showed no physiological or behavioral signs of stress, such as increased heart rate, abnormal behavior, or psychological indices of distress (depressive/anxiety‐related behavior). Agonism was minimal throughout the introduction process and over the subsequent months; only one wound was incurred over the course of the study. Levels of abnormal behaviors, psychological indices of distress, locomotion, inactivity, and affiliation showed improvements within several weeks after introduction; these changes were still present 5–9 months later for the latter two categories. Heart rates during introduction fell significantly in the settled pair phase, and also varied predictably with time of day. Fecal cortisol levels were lower in settled pairs than in single housing. The fact that reductions in abnormal behavior did not persist over the long term may have been confounded by increasing duration of time spent caged. The results of this study may be of practical use for designing and monitoring social introductions and suggest that managers should not dismiss the feasibility of successful pairing of adult male rhesus macaques. Am. J. Primatol. 70:542–550, 2008.

Positive reinforcement training moderates only high levels of abnormal behavior in singly housed rhesus macaques.

This study evaluated the application of positive reinforcement training (PRT) as an intervention for abnormal behaviors in singly housed laboratory rhesus macaques at 2 large primate facilities. Training involved basic control behaviors and body-part presentation. The study compared baseline behavioral data on 30 adult males and 33 adult females compared with 3 treatment phases presented in counterbalanced order: 6 min per week of PRT, 20 or 40 min per week of PRT, and 6 min per week of unstructured human interaction (HI). Within-subject parametric tests detected no main or interaction effects involving experimental phase. However, among a subset of subjects with levels of abnormal in the top quartile of the range (n = 15), abnormal behavior was reduced from 35% to 25% of samples with PRT but not with HI. These results suggest that short durations of PRT applied as enrichment for this species and in this context may not in itself be sufficient intervention for abnormal behavior because levels remained high. However, it may be appropriate as an adjunct to other interventions and may be best targeted to the most severely affected individuals.

Comparing options for pair housing rhesus macaques using behavioral welfare measures

In a biomedical research environment, research or management procedures may render continuous full contact pairing of rhesus macaques (Macaca mulatta) unfeasible. This study aimed to determine whether separation on a frequent basis or housing in adjacent cages with tactile contact interferes with the behavioral benefits of continuous full contact. Behavioral data (1260 hours) were collected from 32 adult females and 16 adult males housed at two National Primate Research Centers. Subjects were studied in four housing conditions: single housing, full contact pair housing, intermittent contact pair housing, and protected contact housing. After introduction, each pair was housed in each of the three social housing conditions in varying order. Among females, but not males, introducing animals into full and intermittent contact reduced levels of abnormal behavior. There was a trend toward this reduction in protected contact. In both females and males, full and intermittent contact was associated with lower levels of anxiety‐related behavior, but protected contact was not. Females spent more time inactive in protected contact than either full or intermittent contact, and males showed a trend toward less inactivity following introduction into full contact. Both sexes showed less affiliation in protected contact compared to the other forms of social housing. Agonistic behavior among females was not affected by housing condition; among males, levels were equivalent in full and intermittent contact but were higher in intermittent than protected contact. Frequent separation of pairs does not appear to detract from the behavioral benefits of pair housing. Separation by a barrier permitting tactile contact is inferior to other forms of social housing but showed modest improvements over single housing nonetheless. This study can guide the provision of social contact to rhesus macaques under conditions restricting pairs from continuous full contact. Am. J. Primatol. 76:30–42, 2014.

Pervasive supply of therapeutic lysosomal enzymes in the CNS of normal and Krabbe‐affected non‐human primates by intracerebral lentiviral gene therapy

Metachromatic leukodystrophy (MLD) and globoid cell leukodystrophy (GLD or Krabbe disease) are severe neurodegenerative lysosomal storage diseases (LSD) caused by arylsulfatase A (ARSA) and galactosylceramidase (GALC) deficiency, respectively. Our previous studies established lentiviral gene therapy (GT) as a rapid and effective intervention to provide pervasive supply of therapeutic lysosomal enzymes in CNS tissues of MLD and GLD mice. Here, we investigated whether this strategy is similarly effective in juvenile non‐human primates (NHP). To provide proof of principle for tolerability and biological efficacy of the strategy, we established a comprehensive study in normal NHP delivering a clinically relevant lentiviral vector encoding for the human ARSA transgene. Then, we injected a lentiviral vector coding for the human GALC transgene in Krabbe‐affected rhesus macaques, evaluating for the first time the therapeutic potential of lentiviral GT in this unique LSD model. We showed favorable safety profile and consistent pattern of LV transduction and enzyme biodistribution in the two models, supporting the robustness of the proposed GT platform. We documented moderate inflammation at the injection sites, mild immune response to vector particles in few treated animals, no indication of immune response against transgenic products, and no molecular evidence of insertional genotoxicity. Efficient gene transfer in neurons, astrocytes, and oligodendrocytes close to the injection sites resulted in robust production and extensive spreading of transgenic enzymes in the whole CNS and in CSF, leading to supraphysiological ARSA activity in normal NHP and close to physiological GALC activity in the Krabbe NHP, in which biological efficacy was associated with preliminary indication of therapeutic benefit. These results support the rationale for the clinical translation of intracerebral lentiviral GT to address CNS pathology in MLD, GLD, and other neurodegenerative LSD.

Enrichment and primate centers: closing the gap between research and practice.

A wealth of published research is available to guide environmental enrichment programs for nonhuman primates, but common practice may not consistently correspond to research findings. A 2003 survey to quantify common practice queried individuals overseeing enrichment programs about (a) social, feeding, structural, and manipulable enrichment; (b) human interaction and training; (c) general program administration; (d) the role of the institutional animal care and use committee (IACUC) in the enrichment program; and (e) the impetus for recent programmatic changes. Returned surveys provided information on the management of 35,863 primates and found social housing significantly more constrained than inanimate enrichment. Survey results suggest that social housing of macaques has not increased significantly over the past decade. The most commonly mentioned constraints related to research protocols. Facilities with thorough IACUC reviews of enrichment issues provided social housing for a significantly larger proportion of primates in biomedical research studies than did those with rare IACUC reviews. IACUC reviews prompted program enhancements much less often than did regulatory or accreditation inspections. These results suggest IACUC review is an underutilized mechanism for improving enrichment programs.

Survey of 2014 behavioral management programs for laboratory primates in the United States

The behavioral management of laboratory nonhuman primates in the United States has not been thoroughly characterized since 2003. This article presents the results of a survey behavioral management programs at 27 facilities and covering a total of 59,636 primates, 27,916 housed in indoor cages and 31,720 in group enclosures. The survey included questions regarding program structure, implementation, and methodology associated with social housing, positive reinforcement training, positive human interaction, exercise enclosures, and several categories of inanimate enrichment. The vast majority of laboratory primates are housed socially (83%). Since 2003, the proportion of indoor‐housed primates reported to be housed singly has fallen considerably, from 59% to 35% in the facilities surveyed. The use of social housing remains significantly constrained by: 1) research protocol requirements, highlighting the value of closely involved IACUCs for harmonizing research and behavioral management; and 2) the unavailability of compatible social partners, underscoring the necessity of objective analysis of the methods used to foster and maintain compatibility. Positive reinforcement training appears to have expanded and is now used at all facilities responding to the survey. The use of enrichment devices has also increased in the participating facilities. For most behavioral management techniques, concerns over the possibility of negative consequences to animals are expressed most frequently for social housing and destructible enrichment, while skepticism regarding efficacy is limited almost exclusively to sensory enrichment. Behavioral management program staffing has expanded over time in the facilities surveyed, due not only to increased numbers of dedicated behavioral management technicians but also to greater involvement of animal care technicians, suggesting an increase in the integration of behavioral care into animal husbandry. Broad awareness of common practice may assist facilities with program evaluation and assessment of progress in the field can generate recommendations for continuing the advancement of primate behavioral management programs. Am. J. Primatol. 78:780–796, 2016.

Astrocyte Atrophy and Immune Dysfunction in Self-Harming Macaques

Background Self-injurious behavior (SIB) is a complex condition that exhibits a spectrum of abnormal neuropsychological and locomotor behaviors. Mechanisms for neuropathogenesis could include irregular immune activation, host soluble factors, and astrocyte dysfunction. Methods We examined the role of astrocytes as modulators of immune function in macaques with SIB. We measured changes in astrocyte morphology and function. Paraffin sections of frontal cortices from rhesus macaques identified with SIB were stained for glial fibrillary acidic protein (GFAP) and Toll-like receptor 2 (TLR2). Morphologic features of astrocytes were determined using computer-assisted camera lucida. Results There was atrophy of white matter astrocyte cell bodies, decreased arbor length in both white and gray matter astrocytes, and decreased bifurcations and tips on astrocytes in animals with SIB. This was combined with a five-fold increase in the proportion of astrocytes immunopositive for TLR2. Conclusions These results provide direct evidence that SIB induces immune activation of astrocytes concomitant with quantifiably different morphology.