archives@tulane.edu / Neutrophils are the most abundant white blood cells in human blood and require continuous replenishment from bone marrow granulopoiesis throughout life. Their function as phagocytes in innate immunity has been well studied, but the dynamics and movement of neutrophils in vivo are less clearly understood. To quantitate the kinetics of neutrophil movement during homeostasis, acute inflammation, and aging, we applied 5-bromo-2'-deoxyuridine (BrdU) pulse-chase labeling followed by hematology and flow cytometry analyses in healthy, acutely-infected, and aged rhesus macaques (Macaca mulatta). We applied our kinetics results to a mathematical model and calculated that neutrophils have a half-life of 1.63 ± 0.16 days and a daily production of 1.42×109 cells / L / day in heathy young adult rhesus macaques. In rhesus macaques undergoing acute inflammation, we followed neutrophil kinetics during acute stages of a bacterial infection (Shigella) and viral infection (SIV). A massive replenishment of neutrophils from bone marrow to blood as indicated by BrdU kinetics occurred as early as 3 days post Shigella inoculation, the degree to which correlated with the Shigella inoculation doses. As animals aged, neutrophil production declined while plasma G-CSF levels increased, and there was an earlier release, as well as higher in-group variability in neutrophil kinetics, particularly upon reaching 20 years of age or older (equivalent to 70 years or more in humans). This suggested a dysregulated feedback mechanism whereby increased levels of G-CSF failed to restore neutrophil production in elderly rhesus macaques that was associated with induced production of pro-inflammatory cytokines and earlier release of less mature neutrophils and PMN-MDSCs. Taken together, we established a rhesus macaque model to study neutrophil kinetics and functions in vivo during homeostasis, acute inflammation, and aging. Our results emphasized the massive production of neutrophils during homeostasis and the continuous requirement but reduced replenishment capabilities during aging. The significance of the results also indicates an important role for these long-discovered leukocytes in maintaining homeostasis beyond phagocytic pathogen clearance. / 1 / Ziyuan He

archives@tulane.edu / Background: Human Immunodeficiency Virus-1 (HIV-1) has killed over 35 million and infects 1.8 million new people each year. Antiretroviral therapy (ART), although effective controlling plasma viremia and transmission, does not purge latent or persistent reservoirs necessary to eliminate infection, and must be maintained for life. It is thus imperative to discover therapeutics that provide both lifetime suppression of viral loads and depletion of viral reservoirs. Methods: To harness the immunosurveillance capacity of highly functional and persistent CMV-specific adaptive response, rhesus PBMCs were stimulated with rhCMV peptide pools (IE1, IE2, and pp65) to expand rhCMV-specific T cells. These cells were then genetically modified with retroviral vectors expressing a CD4 extracellular domain linked to T cell intracellular signaling domains that instruct CTL activity, converting them into HIV-specific effector cells. Vectors combine CD4 targeting with an maC46 fusion inhibitor to protect against viral entry. In a reversal of the critical step in the HIV viral lifecycle whereby virus targets new CD4+ host cells using its Env glycoprotein, these genetic modifications redirect host immune responses to target and kill Env expressing infected cells. We hypothesize that continuous stimulation of CD4-CAR T cells through their rhCMV-specific TCR will maintain activated T effector memory CTL capable of targeting HIV infected cells. Results: We find that autologous rhPBMCs can be expanded ex vivo with rhCMV peptides up to therapeutically relevant numbers for adoptive transfer. This rhCMV-specific T cell expansion enriches cells in a phenotype consistent with T effector memory differentiation. Following genetic modification and adoptive transfer, cells reach peak expansion at seven days post infusion into ART suppressed or unsuppressed SHIV infected Rhesus Macaques. We observe these cells capable of persisting in vivo for at least 2 years following reinfusion. Furthermore, these cells are maintained in vivo in an effector memory phenotype throughout the duration they were analyzed. Despite this, SHIV plasma viral loads remain unchanged. Conclusion: These studies establish use of rhCMV-specific T cells as an effective way to produce persistent genetically modified cells targeting SHIV. Future studies will need to further increase in vivo expansion, protection, and CTL activity as viral loads remain detectable. / 1 / Nathan Michel Johnson

Human and non-human primates (NHP) undergo age-related cognitive decline beginning as early as middle-age, even in the absence of an underlying pathology or disease. Growing evidence indicates that an increase in white mater pathology related to rising chronic levels of inflammation may be key contributors to age related cognitive decline. Curcumin (CUR), the active ingredient in turmeric, is a polyphenol nutraceutical with potent anti-inflammatory and antioxidative effects. Several ongoing research studies are underway to explore this potential anti-aging compound. For the first time in a rhesus monkey model of aging, we studied the effects of CUR supplementation on cognition and inflammation. Baseline MRI, blood, CSF and cognitive data were collected for all monkeys. Monkeys were fed daily doses of 500mg of CUR or a vehicle control over 18-months during which three rounds of a battery of cognitive testing was performed along with regular collection of blood, CSF and MRI. Following completion of this testing and specific to this thesis, monkeys were further tested on object discrimination, object and spatial reversal tasks. No significant differences were observed between groups in object discrimination task performance. CUR treatment improved performance on object reversal testing, with treated monkeys making fewer perseverative type errors. At the completion of behavioral testing, serum samples from two-year post treatment onset and brain tissue were harvested for post-mortem analysis of markers of inflammation. The density and morphology of microglia, the resident immune cells of the brain, were examined using immunohistochemistry on serial coronal sections through frontal cortical gray (A46, A25) and while matter (FWM, CC, and CngB) regions that are implicated in cognitive aging. We demonstrated that CUR treatment did not significantly alter the density of presumably immune-activated microglia expressing the MHC class II marker LN3. However, treatment did affect morphological features of microglia specifically within the while matter. Within the white matter, CUR treatment was associated with a significant increase in microglial ramification, evidenced by greater process length, number of nodes and convex-hull area and volume. Increased microglial ramification suggests greater likelihood of microglial surveillance within the white matter associated with CUR treatment. No significant group differences however were observed in the select serum cytokine levels quantified using multiplex ELISA, or in inflammatory gene expression in brain tissue measured with qRT-PCR. While our findings show the benefit of CUR supplementation on cognitive performance and its effects on microglial morphology, further study is needed to understand the precise changes that CUR supplementation may have on inflammation.

Rhesus macaques are a widely used animal model of human diseases and related immune responses. Fc receptors (FcRs) mediate the interaction between antibody molecules and innate killing mechanisms, consequently eliminating the pathogen. In rhesus macaques, FcRs are highly polymorphic. To evaluate the potential influence of FcgR polymorphisms on the interaction with antibody molecules, we performed in silico analysis using SIFT, Provean, nsSNPAnalyzer, I-Mutant, MuSTAB and iPTREE-STAB web servers. V20G in FcγRI, I137K in FcγRII and I233V in FcγRIII were further analyzed structurally using FOLD-X, AMMP and Chimera to calculate changes in folding and interaction energy and for structure visualization. Results from our analysis suggest that the selected variations destabilize protein structure. Additionally, Q32R increases the binding affinity of FcγRI, whereas A131T decreases the binding affinity of FcγRII towards IgG1. Together, our results indicate that these substitutions might influence effector and regulatory mechanisms resulting from antibody/FcR interactions.

Rhesus macaques represent a valuable model in biomedical research and in development of vaccines and therapeutics. Due to the lack of reagents, the general properties of IgG and corresponding cellular receptors (FcγR) in this species are poorly characterized. We engineered recombinant IgGs containing each of the four rhesus macaque heavy constant region (CH) subclasses. To define FcγRs that mediate IgGs, we identified and characterized three FcγR classes, and generated recombinant cDNA constructs. cDNA IgH constructs were created by fusing – by sequence overlap extension PCRs – a gene segment encoding the murine variable heavy domain specific for the hapten NIP, an established specificity system for assessing antibody effector functions, with rhesus macaque CH fragments. The complete IgH constructs were transfected into J558L cells, a murine IgH-lost myeloma cell line expressing anti-NIP light chain. Secretion of engineered IgGs was determined by ELISAs using NIP-BSA and anti-monkey IgG-specific antibodies. Molecular cloning methods were applied to identify and clone FcγR genes, and recombinant FcγR cDNA constructs were created by the recombinant DNA method. Four engineered IgH cDNA constructs were successfully created. Recombinant IgGs, in the intact Ig form and retaining the original anti-NIP specificity, were successfully produced. Compared to those in humans, FcγRs in rhesus macaques share high homology, yet also feature a relatively high level of intra-species polymorphism and possess different N-linked glycosylation patterns. FcγR constructs and expression vectors were successfully generated. The chimeric recombinant IgGs are powerful tools for defining IgG functional properties and studying CH structure/function relationship. These molecules can also be used as immunogens for generation of antibodies capable of unequivocally detecting individual IgG subclasses. The findings on FcγRs validate rhesus macaques as a model for studying antibody responses, and underscore the need to take into account of the genetic heterogeneity. The FcγR constructs and vectors serve as a tool for further studies of IgG/FcγR interactions. We also reported here our findings from a separate study that the main female hormone, 17β-estradiol, is capable of restoring antibody responses to an influenza vaccine in a postmenopausal mouse model, suggesting that immunogenicity and efficacy of influenza vaccines should be evaluated in postmenopausal women.

<p>The visual world is full of potentially important information, but only a subset of the world can be evaluated at any time. An essential function of the central nervous system is to rapidly and adaptively select which stimuli warrant attention. Much of the time, attention is directed towards stimuli that are relevant for current goals. However, things that have proven important in an organisms' personal or evolutionary past effectively compete with goal-relevant targets for attention. In humans, one example of this attentional superset is faces: faces attract attention even when they are in competition with immediate goals. Using a combination of behavioral, pharmacological, and electrophysiological techniques in the rhesus macaque, I investigated the physiological, neurobiological, and evolutionary determinants of the attentional capture of faces. First, I show that the prioritization of faces is evolutionarily conserved in primates. Face distractors also capture attention in rhesus macaques, a species of old world monkey, successfully competing with task goals for limited attentional resources. Importantly, the same classes of faces have the greatest attentional effects in both monkeys and humans. Further, I describe behavioral evidence that subcortical systems contribute to the attentional salience of faces in this species, proving an initial characterization of the neural mechanisms that may mediate this effect. Next, I examine the interaction between pupil size and vigilance for faces. A focal increase in luminance has long been known to provoke pupil constriction, but here I show that the pupil response to a flashed distractor is proportional to the allocation of attention to that image. Pupil constriction may provide a novel implicit metric of stimulus attention. In particular, face images provoked greater pupil constriction than non-face images. Moreover, I also find that baseline pupil size is a strong predictor of distractor interference, suggesting that arousal may modulate social vigilance. Therefore, I next examined the activity of single neurons within dorsal anterior cingulate cortex (dACC), a region implicated in task performance across a wide variety of tasks, but which also has strong connections to subcortical neuromodulatory centers responsible for regulating arousal. I find that the dACC discriminates between social and nonsocial distractors, scales with distractor attention, and predicts adjustments in arousal and vigilance state on upcoming trials. This is consistent with a model in which dACC supports task performance through regulating arousal. Finally, I turn to oxytocin (OT), a neuromodulatory hormone released during affiliative social interactions that is also implicated in regulating arousal. Though typically thought to generally enhance social attention, I report multiple circumstances in which OT suppresses, rather than enhances, vigilance for faces. This suggests a mechanism through which affiliative social interactions can reduce social vigilance, permitting more relaxed social interactions. Together, these results highlight an evolutionarily conserved neural circuit important for the adaptive, contextual modulation of reflexive face attention, a behavior that is compromised in both anxiety disorders and autism.</p> / Dissertation

abstract: In the past decade, research on the motor control side of neuroprosthetics has steadily gained momentum. However, modern research in prosthetic development supplements a focus on motor control with a concentration on sensory feedback. Simulating sensation is a central issue because without sensory capabilities, the sophistication of the most advanced motor control system fails to reach its full potential. This research is an effort toward the development of sensory feedback specifically for neuroprosthetic hands. The present aim of this work is to understand the processing and representation of cutaneous sensation by evaluating performance and neural activity in somatosensory cortex (SI) during a grasp task. A non-human primate (Macaca mulatta) was trained to reach out and grasp textured instrumented objects with a precision grip. Two different textures for the objects were used, 100% cotton cloth and 60-grade sandpaper, and the target object was presented at two different orientations. Of the 167 cells that were isolated for this experiment, only 42 were recorded while the subject executed a few blocks of successful trials for both textures. These latter cells were used in this study's statistical analysis. Of these, 37 units (88%) exhibited statistically significant task related activity. Twenty-two units (52%) exhibited statistically significant tuning to texture, and 16 units (38%) exhibited statistically significant tuning to posture. Ten of the cells (24%) exhibited statistically significant tuning to both texture and posture. These data suggest that single units in somatosensory cortex can encode multiple phenomena such as texture and posture. However, if this information is to be used to provide sensory feedback for a prosthesis, scientists must learn to further parse cortical activity to discover how to induce specific modalities of sensation. Future experiments should therefore be developed that probe more variables and that more systematically and comprehensively scan somatosensory cortex. This will allow researchers to seek out the existence or non-existence of cortical pockets reserved for certain modalities of sensation, which will be valuable in learning how to later provide appropriate sensory feedback for a prosthesis through cortical stimulation. / Dissertation/Thesis / M.S. Bioengineering 2011

Prenatal stress has been correlated with adverse developmental outcomes affecting infant cognition and behavior. Previous studies have shown that prenatal stress can lead to increased susceptibility to adult disease but few studies have looked at the physiological stress response system by measuring the activity of the hypothalamicpituitary-adrenal (HPA) axis. Cortisol, the output of the HPA axis can be secreted in many different matrices (saliva, blood, urine, feces and hair). Most studies that do, only look at one measure of hormone production instead of examining multiple matrices. Additionally these studies do not look at the relationship between matrices. Hair provides a long-term assessment of cortisol hormone production as related to infant behavior. Four measures of cortisol representative of prenatal and postpartum periods were collected in a sample population of rhesus macaques at the NIH facility. No stress was applied to these animals and cortisol concentrations were assessed in maternal hair, infant hair, amniotic fluid, and mothers’ milk. These cortisol measures were then analyzed first to determine vii the relationships between the four measures and second to relate these cortisol values to infant behavior in the primate neonatal neurobehavioral assessment. Subjects of this study were 30 mothers and infants from the 2015 and 2016 breeding cohort. 25 of which, were unique dyads. Using four statistical analyses and 3 groupings of behavior, we found that maternal hair cortisol concentrations were correlated with different temperaments of infants, while milk cortisol concentrations were correlated with infant’s visual exploration of the environment. Additionally, an inverse relationship was found between hair cortisol concentrations and both hair cortisol concentrations with amniotic fluid cortisol. Together, the four statistical analyses show that Maternal HPA axis activation during and after pregnancy affects infant behavioral development 1 month postpartum.

Numerous studies show that a low level of response to the intoxicating effects of alcohol is considered a risk factor for future alcoholism. However, assessing this sensitivity usually requires administering a controlled dose of alcohol, which has a number of inherent problems. Early observations in our lab suggest that the response to anesthetics that show cross tolerance with alcohol, like ketamine, are blunted in nonhuman primates at risk for high alcohol intake, and may be a viable measure of future alcohol consumption. This study was designed to test potential predictors of future alcohol consumption using the change in ketamine across repeated exposures (i.e., tolerance). In addition, potential mediating factors of alcohol consumption, including early temperament and behavior, were assessed. Subjects were 16 three-year-old, alcohol naïve rhesus macaque males raised by their biological mothers. Ketamine Exposure-Each subject was exposed to three 10.0 mg/kg intramuscular doses of ketamine. The time from injection to recovery from anesthetic was recorded for each dose, to be used as a measure of subject's sensitivity and developed tolerance. Alcohol Intake Assessment-Two weeks after the final ketamine dose, subjects were allowed ad libitum access to a palatable 8.4% alcohol solution for two-hours a day, five days a week, for six weeks. During the Two-Choice phase of testing, subjects were simultaneously given ad libitum access to the 8.4% alcohol solution and to a sweetened solution for two-hours a day, five days a week, for four weeks. Solution consumption was recorded daily and averaged across the weeks for each phase of alcohol testing. Temperament and Behavior-As infants, all subjects participated in a bio-behavioral assessment (BBA), when they were between 90 and 120 days of age. Data collected during the BBA on subjects' temperament (Vigilance, Gentleness, Confidence, and Nervousness) and Behavior (Activity and Emotionality) were used in analyses. Results showed a relationship between the tolerance developed between ketamine doses and average alcohol consumption during the Alcohol-Only phase (r = 0.61, R2 = 0.372, F (1,14) = 8.300, p = 0.012). Average alcohol consumption during the Alcohol-Only phase was also related to ratings of Confidence (r = 0.499, R2=0.249, F(1,14)=4.647, p = 0.049), Activity (Day 1: r = 0.503, R2 = 0.253, F(1,14) = 4.732, p = 0.047; Day 2: r = 0.455, R2 = 0.207, F(1,14) = 3.652, p = 0.077), and Emotionality (r = 0.466, R2 = 0.217, F(1,14) = 3.885, p=0.069). The results of this study suggest that change in ketamine recovery time and early life temperament and behaviors may be measures of future risk for alcohol abuse disorders. This data is limited by the small sample size and future study is necessary to further tease out the relationships between these variables and alcohol consumption.

No description available.