Next-Generation Sequencing-Based Identification of Enterobacter hormaechei as Causative Agent of High Mortality Disease in NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) Mice.

NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice, lacking many components of a mature immune system, are at increased risk of disease. General understanding of potential pathogens of these mice is limited. We describe a high mortality disease outbreak caused by an opportunistic bacterial infection in NSG mice. Affected animals exhibited perianal fecal staining, dehydration, and wasting. Histopathologic lesions included a primary necrotizing enterocolitis, with inflammatory and necrotizing lesions also occurring in the liver, kidneys, heart, and brain of some mice. All affected individuals tested negative for known opportunistic pathogens of immunodeficient mice. We initially identified a member of Enterobacter cloacae complex (ECC) in association with the outbreak by traditional diagnostics. ECC was cultured from extraintestinal organs, both with and without histopathologic lesions, suggesting bacteremia. Infrared spectroscopy and MALDI-TOF mass spectrometry demonstrated that isolates from the outbreak shared molecular features and likely a common origin. We subsequently hypothesized that advanced sequencing methods would identify a single species of ECC associated with clinical disease. Using a novel targeted amplicon-based next-generation sequencing assay, we identified Enterobacter hormaechei in association with this outbreak. Knowledge of this organism as a potential opportunistic pathogen in NSG mice is critical for preclinical studies to prevent loss of animals and confounding of research.

Development of a C3 Humanized Rat as a New Model for Evaluating Novel C3 Inhibitors.

INTRODUCTION: C3 is central for all complement activation pathways, thus making it an attractive therapeutic target. Many C3-targeted agents are under extensive development with one already approved for clinical use. However, most, if not all, C3 inhibitors are human or nonhuman primate C3-specific, making evaluating their efficacies in vivo before a clinical trial extremely difficult and costly. METHODS: We first studied the compatibility of human C3 in the rat complement system, then developed a C3 humanized rat using the CRISPR/Cas9 technology. We thoroughly characterized the resultant human C3 humanized rats and tested the treatment efficacy of an established primate-specific C3 inhibitor in a model of complement-mediated hemolysis in the C3 humanized rats. RESULTS: We found that supplementing human C3 protein into the C3-deficient rat blood restored its complement activity, which was inhibited by rat factor H or compstatin, suggesting that human C3 is compatible to the rat complement system. The newly developed C3 humanized rats appeared healthy and expressed human but not rat C3 without detectable spontaneous C3 activation. More importantly, complement-mediated hemolysis in the C3 humanized rats was also inhibited by compstatin both in vitro and in vivo. CONCLUSION: The successfully developed C3 humanized rats provided a much-desired rodent model to evaluate novel C3 inhibitors in vivo as potential drugs.

Impact of Automated Genotyping and Increased Breeding Oversight on Overall Mouse Breeding Colony Productivity.

Mice have become increasingly popular as genetic tools, facilitated by the production of advanced genetically engineered mouse models (GEMMs). GEMMs often require in-house breeding and production by research groups, which can be quite complex depending on the design of the GEMM. Identification of methods to increase the efficiency of breeding practices offers opportunities to optimize and reduce the number of animals bred for research while maintaining similar research output. We investigated the use of commercial automated genotyping and centralized breeding management on overall breeding colony productivity in a colony of multiple GEMM lines. This study involved a three-group study design, where the first group continued their standard breeding practices (group A), the second utilized standard breeding practices but outsourced genotyping in place of inhouse genotyping (group B), and a third group outsourced genotyping and had assistance with routine breeding practices from the laboratory animal care team (group C). Compared to standard practice (group A), groups B and C produced more cages and mice over time, which appeared to be driven primarily by an increase in the number of breeding cages in each colony. Higher numbers of breeders correlated with an increased number of litters and generation of new cages. The increases in colony productivity measures were further enhanced in group C compared to group B. The overall cost associated with producing new animals was lowest in group B, followed by groups A and C. Although, by the end of the study, cost to produce new mice was comparable between all three groups. These data suggest that by optimizing breeding practices and management, fewer animals could be utilized to produce the same amount of progeny and reduce overall animal usage and production.

Elucidation of the Central Serotonin Metabolism Pathway in Rhesus Macaques (Macaca mulatta) with Self-injurious Behavior.

Macaques with self-injurious behavior (SIB) have been used as a model of human SIB and have previously been shown to respond to treatments targeting enhancement of central serotonin signaling, whether by supplementation with tryptophan, or by inhibiting synaptic reuptake. Decreased serotonin signaling in the brain has also been implicated in many human psychopathologies including major depression disorder. A disturbance in tryptophan metabolism that moves away from the production of serotonin and toward the production of kynurenine has been proposed as a major etiological factor of depression. We hypothesized that in macaques with SIB, central tryptophan metabolism would be shifted toward kynurenine production, leading to lower central serotonin (5-hydroxytryptamine). We analyzed tryptophan metabolites in the cerebral spinal fluid (CSF) of macaques with and without SIB to determine whether and where tryptophan metabolism is altered in affected animals as compared with behaviorally normal controls. We found that macaques with SIB had lower CSF concentrations of serotonin than did behaviorally normal macaques, and that these deficits were inversely correlated with the severity of abnormal behavior. However, our results suggest that this decrease is not due to shifting of the tryptophan metabolic pathway toward kynurenine, as concentrations of kynurenine were also low. Concentrations of IL6 were elevated, suggesting central inflammation. Determining the mechanism by which serotonin function is altered in self-injurious macaques could shed light on novel therapies for SIB and other disorders of serotonin signaling.

Effects of Pair Housing on Patency of Jugular Catheters in Rats (Rattus norvegicus).

Chronic vascular access devices are widely used in a variety of species for repeated blood sampling or substance administration. Jugular catheters are commonly used for studying addiction-related behaviors in rats. Rats with catheters have historically been individually housed for the duration of the study to prevent cage mates from damaging the catheter. The 2 goals of this study were to determine 1) the effects of pair housing on catheter patency and 2) the effects of pair housing on catheter patency of rats in a study of opioid self-administration and cue-induced reinstatement of opioid-seeking behavior. The latter study also represented an opportunity for experimental refinement as it evaluated the temporary use of a barrier that allowed for pair-housed rats to be physically separated. Male Heterogeneous Stock (HS; n = 24) and Sprague-Dawley (SD; n = 121) rats were allocated to either single- or pair-housed condition. To assess the effect of social housing on catheter patency, rats (HS, n = 24; SD, n = 36) were monitored in their assigned housing condition for one month, with scheduled evaluation of catheter patency and structural damage. To examine the effect of social housing on catheter patency during a study of opioid self-administration and cue-induced reinstatement of opioid-seeking behavior, rats (SD, n = 85) were monitored in their assigned housing condition with similar routine patency evaluations. Catheter patency rates between single- and pairhoused rats were not statistically different in the first experiment, and pair-housed animals were successfully maintained on an infusion study in the second experiment. The use of a barrier between pair-housed rats after surgery allowed continued social contact with no observed adverse effects. These results suggest that, pair housing is a viable option for rats with chronic vascular implants, and may improve their wellbeing by allowing them to display species-typical social behaviors.

Effects of Compressed Paper Bedding on Mouse Breeding Performance and Recognition of Animal Health Concerns.

The combination of bedding substrate and nesting material within the microenvironment of mice is an important consideration for animal care programs in regard to optimizing animal wellbeing. We used 3 general or breeding mouse colonies in our institution to evaluate the effects of bedding substrate on nest building, breeding performance, and recognition of animal health concerns. A scoring system was developed to assess the incorporation of bedding into the nest cup base and walls (nest base incorporation, NBI) in a controlled study with mice bedded on either compressed paper (CP) or corncob (CC) bedding. Compared with CC cages, CP cages had higher NBI scores. To determine the influence of bedding type on the recognition of animal health concerns in an animal facility, cages bedded with CC followed by CP were evaluated for the overall frequency of health-concern reports during a 2-mo time frame for each bedding type in a single-subject A-B study design. The frequency of animal health-concern reports was similar in cages using CC or CP bedding. The animal health condition, rather than bedding type, was associated with the severity of the health problem at the initial report. Breeding performance was compared for 6 mo in matched CC and CP cages containing one of 13 genetically modified mouse lines. NBI scores were higher for breeders housed on CP compared with CC bedding. Monogamous breeder pairs housed on CP had significantly higher indexes of breeding performance (measured as the number of pups per dam per week on study) than did CC cages. This report supports the use of CP bedding in the mouse microenvironment to improve general wellbeing by supporting nesting behavior and reproductive performance without hindering the detection of animal health concerns.

Development of Mast Cell and Eosinophil Hyperplasia and HLH/MAS-Like Disease in NSG-SGM3 Mice Receiving Human CD34+ Hematopoietic Stem Cells or Patient-Derived Leukemia Xenografts.

Immunocompromised mouse strains expressing human transgenes are being increasingly used in biomedical research. The genetic modifications in these mice cause various cellular responses, resulting in histologic features unique to each strain. The NSG-SGM3 mouse strain is similar to the commonly used NSG (NOD scid gamma) strain but expresses human transgenes encoding stem cell factor (also known as KIT ligand), granulocyte-macrophage colony-stimulating factor, and interleukin 3. This report describes 3 histopathologic features seen in these mice when they are unmanipulated or after transplantation with human CD34+ hematopoietic stem cells (HSCs), virally transduced hCD34+ HSCs, or a leukemia patient-derived xenograft. The first feature is mast cell hyperplasia: unmanipulated, naïve mice develop periductular pancreatic aggregates of murine mast cells, whereas mice given the aforementioned human cells develop a proliferative infiltrative interstitial pancreatic mast cell hyperplasia but with human mast cells. The second feature is the predisposition of NSG-SGM3 mice given these human cells to develop eosinophil hyperplasia. The third feature, secondary hemophagocytic lymphohistiocytosis/macrophage activation syndrome (HLH/MAS)-like disease, is the most pronounced in both its clinical and histopathologic presentations. As part of this disease, a small number of mice also have histiocytic infiltration of the brain and spinal cord with subsequent neurologic or vestibular signs. The presence of any of these features can confound accurate histopathologic interpretation; therefore, it is important to recognize them as strain characteristics and to differentiate them from what may be experimentally induced in the model being studied.

Compressed Paper as an Alternative to Corn Cob Bedding in Mouse (Mus musculus) Cages.

Bedding material is a critical component of the mouse environment and affects animal wellbeing and research integrity. Corn cob (CC) bedding has been a common bedding choice in research despite several potential negative aspects of its use. We investigated the use of compressed paper (CP) bedding as a refinement to CC bedding. CP bedding demonstrated greater total and immediate absorption, compared with CC bedding. CP-bedded cages had a reduced frequency of early cage changing prior to the Guide-recommended 2-wk interval for IVC; this reduction was proportional to room census. Intracage ammonia levels were lower in CP-bedded IVC compared with CC-bedded IVC, independent of the age, sex, and number of mice per cage. By contrast, ammonia levels were similar between CP-bedded and CC-bedded static cages. Collectively, these data support the use of CP bedding as a refinement for CC in ventilated mouse cages, in light of increased husbandry efficiency and its positive effect on the welfare of mice.

A conserved intratumoral regulatory T cell signature identifies 4-1BB as a pan-cancer target.

Despite advancements in targeting the immune checkpoints program cell death protein 1 (PD-1), programmed death ligand 1 (PD-L1), and cytotoxic T lymphocyte-associated protein 4 (CTLA-4) for cancer immunotherapy, a large number of patients and cancer types remain unresponsive. Current immunotherapies focus on modulating an antitumor immune response by directly or indirectly expanding antitumor CD8 T cells. A complementary strategy might involve inhibition of Tregs that otherwise suppress antitumor immune responses. Here, we sought to identify functional immune molecules preferentially expressed on tumor-infiltrating Tregs. Using genome-wide RNA-Seq analysis of purified Tregs sorted from multiple human cancer types, we identified a conserved Treg immune checkpoint signature. Using immunocompetent murine tumor models, we found that antibody-mediated depletion of 4-1BB-expressing cells (4-1BB is also known as TNFRSF9 or CD137) decreased tumor growth without negatively affecting CD8 T cell function. Furthermore, we found that the immune checkpoint 4-1BB had a high selectivity for human tumor Tregs and was associated with worse survival outcomes in patients with multiple tumor types. Thus, antibody-mediated depletion of 4-1BB-expressing Tregs represents a strategy with potential activity across cancer types.

Enrichment Preferences of Singly Housed Zebrafish (Danio rerio).

Despite the increasing popularity of zebrafish (Danio rerio) as an animal model, the environmental enrichment preferences of this species have been largely unexplored. We sought to determine the preferences of mature female zebrafish that were singly housed with or without access to one of 10 inanimate forms of enrichment. As a marker of preference, in-tank fish location was observed by video recording. All subjects showed a preference for the front of the tank when caretakers entered the room, demonstrating an effect of human presence on tank location. Among the 10 enrichment items tested, subjects showed the strongest preference for mirrored paper on the side of the tank when compared with the barren half of the tank. Fish also were observed interacting with PVC pipe, marbles, and tulle. Given the preference for enrichment imitating social interaction, we conducted a second study to assess the value of visual exposure of conspecifics in adjacent tanks. The experimental zebrafish were then provided one of 3 conditions-a singly housed neighbor fish, group-housed neighbor fish, or no neighbor fish. All zebrafish housed next to neighboring fish showed a preference to be on the side of the tank nearer to the other fish. Overall, our data indicate that singly housed zebrafish prefer enrichment items that resemble or promote social behaviors. Therefore items such as mirrored paper or housing next to conspecifics should be strongly considered as enrichment strategies for singly housed zebrafish.

Murine Oviductal High-Grade Serous Carcinomas Mirror the Genomic Alterations, Gene Expression Profiles, and Immune Microenvironment of Their Human Counterparts.

Robust preclinical models of ovarian high-grade serous carcinoma (HGSC) are needed to advance our understanding of HGSC pathogenesis and to test novel strategies aimed at improving clinical outcomes for women with the disease. Genetically engineered mouse models of HGSC recapitulating the likely cell of origin (fallopian tube), underlying genetic defects, histology, and biologic behavior of human HGSCs have been developed. However, the degree to which the mouse tumors acquire the somatic genomic changes, gene expression profiles, and immune microenvironment that characterize human HGSCs remains unclear. We used integrated molecular characterization of oviductal HGSCs arising in the context of Brca1, Trp53, Rb1, and Nf1 (BPRN) inactivation to determine whether the mouse tumors recapitulate human HGSCs across multiple domains of molecular features. Targeted DNA sequencing showed the mouse BPRN tumors, but not endometrioid carcinoma-like tumors based on different genetic defects (e.g., Apc and Pten), acquire somatic mutations and widespread copy number alterations similar to those observed in human HGSCs. RNA sequencing showed the mouse HGSCs most closely resemble the so-called immunoreactive and mesenchymal subsets of human HGSCs. A combined immuno-genomic analysis demonstrated the immune microenvironment of BPRN tumors models key aspects of tumor-immune dynamics in the immunoreactive and mesenchymal subtypes of human HGSC, with enrichment of immunosuppressive cell subsets such as myeloid-derived suppressor cells and regulatory T cells. The findings further validate the BPRN model as a robust preclinical experimental platform to address current barriers to improved prevention, diagnosis, and treatment of this often lethal cancer. SIGNIFICANCE: The acquired gene mutations, broad genomic alterations, and gene expression and immune cell-tumor axis changes in a mouse model of oviductal serous carcinoma closely mirror those of human tubo-ovarian high-grade serous carcinoma.

Immunogenicity of prostate cancer is augmented by BET bromodomain inhibition.

BACKGROUND: Prostate cancer responds poorly to current immunotherapies. Epigenetic therapies such as BET Bromodomain inhibition can change the transcriptome of tumor cells, possibly making them more immunogenic and thus susceptible to immune targeting. METHODS: We characterized the effects of BET bromodomain inhibition using JQ1 on PD-L1 and HLA-ABC expression in two human prostate cell lines, DU145 and PC3. RNA-Seq was performed to assess changes on a genome-wide level. A cytotoxic T cell killing assay was performed in MC38-OVA cells treated with JQ1 to demonstrate increased immunogenicity. In vivo experiments in the Myc-Cap model were conducted to show the effects of JQ1 administration in concert with anti-CTLA-4 checkpoint blockade. RESULTS: Here, we show that targeting BET bromodomains using the small molecule inhibitor JQ1 decreased PD-L1 expression and mitigated tumor progression in prostate cancer models. Mechanistically, BET bromodomain inhibition increased MHC I expression and increased the immunogenicity of tumor cells. Transcriptional profiling showed that BET bromodomain inhibition regulates distinct networks of antigen processing and immune checkpoint molecules. In murine models, treatment with JQ1 was additive with anti-CTLA-4 immunotherapy, resulting in an increased CD8/Treg ratio. CONCLUSIONS: BET Bromodomain inhibition can mediate changes in expression at a genome wide level in prostate cancer cells, resulting in an increased susceptibility to CD8 T cell targeting. These data suggest that combining BET bromodomain inhibition with immune checkpoint blockade may have clinical activity in prostate cancer patients.

Variation in Bacterial Contamination of Microisolation Cage Tops According to Rodent Species and Housing System.

The Guide recommends sanitizing cage components, including microisolation cage tops (MCT), at a minimum of every 2 wk. Previously published data demonstrated that mouse MCT microbial loads do not increase until at least 2 wk and that sanitation can be delayed past 2 wk. How microbial loads differ on mouse compared with rat MCT, as well as across different ventilation systems, remains unclear. We hypothesized that MCT microbial loads would be higher in tops from rats compared with mice and would differ according to IVC ventilation system. We evaluated bacterial loads on MCT at serial time points to 90 d from static cages housing mice or rats and from rat and mouse cages on several ventilation systems (mice, 6; rats, 4). MCT were determined to have sufficiently elevated bacterial loads to necessitate changing based on either statistically significant changes in bacterial loads or values greater than 50 cfu. Across all ventilation systems, bacterial counts at 14 d were significantly higher on rat MCT compared with mouse MCT. Across the ventilation systems examined, rat MCT cfu remained similarly elevated from 14 d through 90 d. Mouse MCT total cfu were also stable across multiple ventilation systems yet remained lower than 50 cfu until at least 90 d. Patterns of bacterial species isolated from rat MCT were relatively consistent over time and ventilation system, whereas mice showed greater variability in both contexts. We found that 14 d is an appropriate sanitization time point for rat MCT, whereas the interval at which mouse MCT are cleaned can be extended to 90 d at least. Our data highlight interspecies differences in the accumulation of bacteria on MCT and that mouse MCT sanitation intervals for several housing systems can be extended beyond 14 d.

Effectiveness of Various Floor Contamination Control Methods in Reducing Environmental Organic Load and Maintaining Colony Health in Rodent Facilities.

Floor contamination control practices in rodent housing facilities commonly include disposable shoe covers despite the lack of evidence for their usefulness in bioexclusion. Contamination control flooring mats are advertised as an economical and environmentally-responsible alternative to shoe covers, yet little is published regarding their efficacy in preventing the transfer of organic material and the introduction of infectious agents into facilities. We evaluated 4 floor contamination control strategies-shoe covers (ShCv), contamination control flooring (CCF), using both products concurrently (ShCv+CCF) compared with using neither-in preventing bacterial transfer and reducing organic load on facility floors and maintaining murine colony health status. According to PCR assay and culture analysis, ShCv provided the greatest reduction in bacte- rial numbers. Either ShCv, CCF, or ShCv+CCF significantly decreased ATP levels within the facility compared with those at facility entrances, with ShCv+CCF yielding the greatest reduction; however, even when neither ShCv nor CCF was used, intrafacility floor ATP levels were about half those at entrances. According to PCR analyses, no murine parasitic, viral, and bacterial pathogens excluded at the institution were detected in any floor, exhaust air dust, or sentinel samples at any time or location, regardless of the floor contamination control method in use. These findings show that floor contamination control methods help to reduce the organic load in rodent IVC facilities but do not enhance protection from environmental contamination due to murine pathogens.

The Endothelin-A Receptor Antagonist Zibotentan Induces Damage to the Nasal Olfactory Epithelium Possibly Mediated in Part through Type 2 Innate Lymphoid Cells.

Zibotentan, an endothelin-A receptor antagonist, has been used in the treatment of various cardiovascular disorders and neoplasia. Castrated athymic nude mice receiving zibotentan for a preclinical xenograft efficacy study experienced weight loss, gastrointestinal bloat, and the presence of an audible respiratory click. Human side effects have been reported in the nasal cavity, so we hypothesized that the nasal cavity is a target for toxicity in mice receiving zibotentan. Lesions in the nasal cavity predominantly targeted olfactory epithelium in treated mice and were more pronounced in castrated animals. Minimal lesions were present in vehicle control animals, which suggested possible gavage-related reflux injury. The incidence, distribution, and morphology of lesions suggested direct exposure to the nasal mucosa and a possible systemic effect targeting the olfactory epithelium, driven by a type 2 immune response, with group 2 innate lymphoid cell involvement. Severe nasal lesions may have resulted in recurrent upper airway obstruction, leading to aerophagia and associated clinical morbidity. These data show the nasal cavity is a target of zibotentan when given by gavage in athymic nude mice, and such unanticipated and off-target effects could impact interpretation of research results and animal health in preclinical studies.

HIV-antibody complexes enhance production of type I interferon by plasmacytoid dendritic cells.

Type I IFN production is essential for innate control of acute viral infection; however, prolonged high-level IFN production is associated with chronic immune activation in HIV-infected individuals. Although plasmacytoid DCs (pDCs) are a primary source of IFN, the mechanisms that regulate IFN levels following the acute phase are unknown. We hypothesized that HIV-specific Ab responses regulate late IFN production. We evaluated the mechanism through which HIV-activated pDCs produce IFN as well as how both monoclonal HIV-specific Abs and Abs produced in natural HIV infection modulated normal pDC sensing of HIV. We found that HIV-induced IFN production required TLR7 signaling, receptor-mediated entry, fusion, and viral uncoating, but not endocytosis or HIV life cycle stages after uncoating. Abs directed against the HIV envelope that do not interfere with CD4 binding markedly enhanced the IFN response, irrespective of their ability to neutralize CD4+ T cell infection. Ab-mediated enhancement of IFN production required Fc γ receptor engagement, bypassed fusion, and initiated signaling through both TLR7 and TLR9, which was not utilized in the absence of Ab. Polyclonal Abs isolated from HIV-infected subjects also enhanced pDC production of IFN in response to HIV. Our data provide an explanation for high levels of IFN production and immune activation in chronic HIV infection.

TGFß1-Mediated SMAD3 Enhances PD-1 Expression on Antigen-Specific T Cells in Cancer.

Programmed death-1 (PD-1) is a coinhibitory receptor that downregulates the activity of tumor-infiltrating lymphocytes (TIL) in cancer and of virus-specific T cells in chronic infection. The molecular mechanisms driving high PD-1 expression on TILs have not been fully investigated. We demonstrate that TGFß1 enhances antigen-induced PD-1 expression through SMAD3-dependent, SMAD2-independent transcriptional activation in T cells in vitro and in TILs in vivo The PD-1hi subset seen in CD8+ TILs is absent in Smad3-deficient tumor-specific CD8+ TILs, resulting in enhanced cytokine production by TILs and in draining lymph nodes and antitumor activity. In addition to TGFß1's previously known effects on T-cell function, our findings suggest that TGFß1 mediates T-cell suppression via PD-1 upregulation in the tumor microenvironment (TME). They highlight bidirectional cross-talk between effector TILs and TGFß-producing cells that upregulates multiple components of the PD-1 signaling pathway to inhibit antitumor immunity. SIGNIFICANCE: Engagement of the coinhibitory receptor PD-1 or its ligand, PD-L1, dramatically inhibits the antitumor function of TILs within the TME. Our findings represent a novel immunosuppressive function of TGFß and demonstrate that TGFß1 allows tumors to evade host immune responses in part through enhanced SMAD3-mediated PD-1 expression on TILs. Cancer Discov; 6(12); 1366-81. ©2016 AACRThis article is highlighted in the In This Issue feature, p. 1293.

Severity and Distribution of Wounds in Rhesus Macaques (Macaca mulatta) Correlate with Observed Self-Injurious Behavior.

Self-injurious behavior (SIB) occurs within laboratory-housed NHP at low frequency but can have a devastating effect on animal research and wellbeing. One barrier to the study and clinical management of these cases is the cost of equipment and personnel time to quantify the behavior according to the current standard of observation and to score remotely obtained video recordings. In studies of human SIB, in which direct observation is difficult or prohibited, researchers have demonstrated that quantifying the tissue damage resulting from SIB can be a useful proxy to represent the underlying behavior. We hypothesized that the nature of wounds resulting from SIB in NHP could be used in a similar manner to measure the abnormal behavior. Using a cohort of rhesus macaques with high-incidence SIB, we examined severity, distribution, and number of wounds and compared them with observed incidences of SIB during a 12-wk experiment. We found that the number, severity, and distribution of physical wounds were associated with the incidences of biting behavior observed during the 2 wk prior to measurement. We also found that an increased number of wounds was associated with increased severity. Animals with wounds of moderate severity were more likely to also have severe wounds than were macaques with wounds that were lower than moderate in severity. This work is the first representative study in NHP to find that behavioral SIB correlates with physical wounding and that increases in the frequency and number of the body regions affected correlates with the severity of wounding.