Juvenile Rhesus Research Articles

Inner Retinal Microvasculature With Refraction in Juvenile Rhesus Monkeys.

To characterize inner retinal microvasculature of rhesus monkeys with a range of refractive errors using optical coherence tomography angiography. Refractive error was induced in right eyes of 18 rhesus monkeys. At 327 to 347days of age, axial length and spherical equivalent refraction (SER) were measured, and optical coherence tomography and optical coherence tomography angiography scans (Spectralis, Heidelberg) were collected. Magnification-corrected metrics included foveal avascular zone area and perfusion density, fractal dimension, and lacunarity of the superficial vascular complex (SVC) and deep vascular complex (DVC) in the central 1-mm diameter and 1.0- to 1.5-mm, 1.5- to 2.0-mm, and 2.0- to 2.5-mm annuli. Pearson correlations were used to explore relationships. The mean SER and axial length were 0.78 ± 4.02 D (-7.12 to +7.13 D) and 17.96 ± 1.08mm (16.41 to 19.93mm), respectively. The foveal avascular zone area and SVC perfusion density were correlated with retinal thickness for the central 1mm (P < 0.05). SVC perfusion density of 2.0- to 2.5-mm annulus decreased with increasing axial length (P < 0.001). SVC and DVC fractal dimensions of 2.0- to 2.5-mm were correlated with axial length and SER, and DVC lacunarity of 1.5- to 2.0-mm annulus was correlated with axial length (P < 0.05). Several inner retinal microvasculature parameters were associated with increasing axial length and SER in juvenile rhesus monkeys. These findings suggest that changes in retinal microvasculature could be indicators of refractive error development. In juvenile rhesus monkeys, increasing myopic refraction and axial length are associated with alterations in the inner retinal microvasculature, which may have implications in myopia-related changes in humans.

Prenatal Zika virus exposure is associated with lateral geniculate nucleus abnormalities in juvenile rhesus macaques.

Zika virus' neural tropism causes significant neural pathology, particularly in developing fetuses. One of the consistent findings from humans and animal models is that prenatal exposure to Zika virus (ZIKV) causes pathology in the eyes and visual pathways of the brain, although the extent to which this pathology persists over development is not clear. In the present report, we build upon our previous work which demonstrated that full-term rhesus monkey ( Macaca mulatta ) fetuses who were exposed to ZIKV early in gestation had significant pathological abnormalities to the organization of the lateral geniculate nucleus (LGN), a major hub of the visual network. The objective of the present work was to replicate those LGN findings and determine whether such pathology persisted across childhood development. We carried out histological analyses of the LGNs of two juvenile rhesus monkeys who were prenatally exposed to ZIKV and two age-matched controls. Pregnant rhesus monkeys were infected with ZIKV via the intravenous and intra-amniotic routes and tracked across development. Following sacrifice and perfusion, brains were subjected to quantitative neuroanatomical analyses with a focus on the size and structure of the LGN and its composite layers. Early fetal ZIKV exposure resulted in developmental abnormalities within the brains' visual pathway: specifically disorganization, blending of layers, laminar discontinuities, and regions of low cell density within the LGN. These abnormalities were not observed in the control animals. Our findings demonstrate that the ZIKV's damage to the LGN that occurs during fetal development persists into childhood.

Bone Marrow Mesenchymal Stem Cells Reversed Ovarian Aging-related m6A RNA Methylation Modification Profile in Aged Granulosa Cells

BackgroundOvarian ageing causes endocrine disturbances and the degeneration of systemic tissue and organ functions to seriously affect women's physical and mental health, and effective treatment methods are urgently needed. Based on our previous studies using juvenile rhesus monkey bone marrow mesenchymal stem cells (BMMSCs) to treat ovarian ageing in rhesus monkey, we found that BMMSCs improved ovarian structure and function. This study continues to explore the mechanism by which BMMSCs reversed granulosa cell (GC) ageing.MethodsA GC ageing model and coculture system of BMMSCs were established, changes in the level of the N6-methyladenosine (m6A) methylation modification were detected, m6A-modified RNA immunoprecipitation sequencing (MeRIP-seq) were performed, correlations between m6A peaks and mRNA expression were determined, and the expression of hub genes was identified using Q-PCR, immunofluorescence staining, and western blot.ResultsOur results showed that H2O2 successfully induced GC ageing and that BMMSCs reversed measures of GC ageing. BMMSCs increased the expression of the FTO protein and reduced the overall level of m6A. We identified 797 m6A peaks (348 hypomethylated and 449 hypermethylated peaks) and 817 differentially expressed genes (DEGs) (412 upregulated and 405 downregulated) after aged GCs were cocultured with BMMSCs, which significantly associated with ovarian function and epigenetic modification. The epigenetic repressive mark and important cell cycle regulator lysine demethylase 8 (KDM8) was downregulated at both the mRNA and protein levels, histone H3 was upregulated in aged GCs after BMMSC coculture, and KDM8 was upregulated after FTO was inhibited through FB23.ConclusionsOur study revealed an essential role for m6A in BMMSCs in reversing GC ageing, and FTO regulated KDM8 mediates histone H3 changes may as a novel regulatory mechanism in BMMSCs to reverse GC ageing.Graphical

Delineation of biomarkers and molecular pathways of residual effects of fluoxetine treatment in juvenile rhesus monkeys by proteomic profiling.

Fluoxetine (Prozac™) is the only antidepressant approved by the US Food and Drug Administration (FDA) for the treatment of major depressive disorder (MDD) in children. Despite its considerable efficacy as a selective serotonin reuptake inhibitor, the possible long-term effects of fluoxetine on brain development in children are poorly understood. In the current study, we aimed to delineate molecular mechanisms and protein biomarkers in the brains of juvenile rhesus macaques (Macaca mulatta) one year after the discontinuation of fluoxetine treatment using proteomic and phosphoproteomic profiling. We identified several differences in protein expression and phosphorylation in the dorsolateral prefrontal cortex (DLPFC) and cingulate cortex (CC) that correlated with impulsivity in animals, suggesting that the GABAergic synapse pathway may be affected by fluoxetine treatment. Biomarkers in combination with the identified pathways contribute to a better understanding of the mechanisms underlying the chronic effects of fluoxetine after discontinuation in children.

Variation in Hypothalamic GnIH Expression and Its Association with GnRH and Kiss1 during Pubertal Progression in Male Rhesus Monkeys (Macaca mulatta).

Modulation of pulsatile gonadotropin-releasing hormone (GnRH) secretion across postnatal development in higher primates is not fully understood. While gonadotropin-inhibitory hormone (GnIH) is reported to suppress reproductive axis activity in birds and rodents, little is known about the developmental trajectory of GnIH expression in rhesus monkeys throughout the pubertal transition. This study was aimed at examining the variation in GnIH immunoreactivity (-ir) and associated changes among GnIH, GnRH, and Kiss1 mRNA expression in the hypothalamus of infant, juvenile, prepubertal, and adult male rhesus monkeys. The brains from rhesus macaques were collected from infancy until adulthood and were examined using immunofluorescence and RT-qPCR. The mean GnIH-ir was found to be significantly higher in prepubertal animals (p &lt; 0.01) compared to infants, and significantly reduced in adults (p &lt; 0.001). Significantly higher (p &lt; 0.001) GnRH and Kiss1 mRNA expression was noted in adults while GnIH mRNA expression was the highest at the prepubertal stage (p &lt; 0.001). Significant negative correlations were seen between GnIH-GnRH (p &lt; 0.01) and GnIH-Kiss1 (p &lt; 0.001) expression. Our findings suggest a role for GnIH in the prepubertal suppression of the reproductive axis, with disinhibition of the adult reproductive axis occurring through decreases in GnIH. This pattern of expression suggests that GnIH may be a viable target for the development of novel therapeutics and contraceptives for humans.

Effects of social rank and pubertal delay on brain structure in female rhesus macaques

Adverse social experience during childhood and adolescence leads to developmental alterations in emotional and stress regulation and underlying neurocircuits. We examined the consequences of social subordination (low social rank) in juvenile female rhesus monkeys, as an ethologically valid model of chronic social stressor exposure, on brain structural and behavioral development through the pubertal transition. Adolescence is a developmental period of extensive brain remodeling and increased emotional and stress reactivity. Puberty-induced increases in gonadal hormones, particularly estradiol (E2), are likely involved due to its organizational effects on the brain and behavior. Thus, we also examined how experimentally delaying pubertal onset with Lupron (gonadotropin releasing hormone -GnRH- agonist used clinically to delay early puberty) interacted with social rank (dominant vs. subordinate) to affect brain and behavioral outcomes. Using a longitudinal experimental design, structural MRI (sMRI) scans were collected on socially housed juvenile female rhesus monkeys living in indoor-outdoor enclosures prior to the onset of puberty (18–25 months), defined as menarche or the initial occurrence of perineal swelling and coloration, and again at 29–36 months, when all control animals had reached puberty but none of the Lupron-treated had. We examined the effects of both social rank and pubertal delay on overall structural brain volume (i.e. intracranial, grey matter (GM) and white matter (WM) volumes), as well as on cortico-limbic regions involved in emotion and stress regulation: amygdala (AMYG), hippocampus (HC), and prefrontal cortex (PFC). Measures of stress physiology, social behavior, and emotional reactivity were collected to examine functional correlates of the brain structural effects. Apart from expected developmental effects, subordinates had bigger AMYG volumes than dominant animals, most notably in the right hemisphere, but pubertal delay with Lupron-treatment abolished those differences, suggesting a role of gonadal hormones potentiating the brain structural impact of social stress. Subordinates also had elevated baseline cortisol, indicating activation of stress systems. In general, Lupron-treated subjects had smaller AMYG and HC volume than controls, but larger total PFC (driven by bigger GM volumes), and different, region-specific, developmental patterns dependent on age and social rank. These findings highlight a region-specific effect of E2 on structural development during female adolescence, independent of those due to chronological age. Pubertal delay and AMYG volume, in turn, predicted differences in emotional reactivity and social behavior. These findings suggest that exposure to developmental increases in E2 modifies the consequences of adverse social experience on the volume of cortico-limbic regions involved in emotional and stress regulation during maturation. But, even more importantly, they indicate different brain structural effects of chronological age and pubertal developmental stage in females, which are very difficult to disentangle in human studies. These findings have additional relevance for young girls who experience prolonged pubertal delays or for those whose puberty is clinically arrested by pharmacological administration of Lupron.

Inverse age-related changes between hypothalamic NPY and KISS1 gene expression during pubertal initiation in male rhesus monkey

The neuroendocrine mechanism underlying the sinusoidal wave nature of gonadotropin–releasing hormone pulse generator activity from infantile to adult age still needs to be meticulously defined. Direct inhibition of kisspeptin neurons by neuropeptide Y (NPY) and close intimacy between the two rekindle the importance of these two neuropeptides controlling reproductive axis activity. Thus, the present study was undertaken to decipher simultaneous fluctuations and to profile correlative changes in the relative expression of KISS1, NPY, and their receptor genes from the mediobasal hypothalamus of infant (n = 3), juvenile, pre-pubertal, and adult (n = 4 in each stage) male rhesus monkey (Macaca mulatta) by RT-qPCR. Significant elevation (p < 0.05−0.01) in KISS1 and KISS1R and low (p < 0.05) expression in NPY and NPY1R mRNA in the adult group as compared to the pre-pubertal group was observed. Moreover, significantly high (p < 0.05) expression of NPY and NPY1R mRNA with non-significant (p> 0.05) decline in KISS1 and KISS1R in pre-pubertal animals in comparison to infants describe inverse correlative age-associated changes during pubertal development. Current findings imply that NPY may contribute as a neurobiological brake for the dormancy of kisspeptin neurons before pubertal onset, while dwindling of this brake is likely to occasion kisspeptin dependent hypothalamic–pituitary–gonadal axis activation at puberty. These findings may help in the development of clinical and therapeutic strategies to regulate fertility in humans.

The behavioral neuroendocrinology of dopamine systems in differently reared juvenile male rhesus monkeys (Macaca mulatta)

Dopamine (DA) is a critical neuromodulator of behavior. With propensities for addiction, hyper-activity, cognitive impairment, aggression, and social subordinance, monkeys enduring early maternal deprivation evoke human disorders involving dopaminergic dysfunction. To examine whether DA system alterations shape the behavioral correlates of adverse rearing, male monkeys (Macaca mulatta) were either mother-reared (MR: N = 6), or separated from their mothers at birth and nursery-reared (NR: N = 6). Behavior was assessed during 20-minute observations of subjects interacting with same- or differently-reared peers. Cerebrospinal fluid (CSF) biogenic amines, and serum testosterone (T), cortisol (CORT), and prolactin (PRL) were collected before and after pharmacologic challenge with saline or the DA receptor-2 (DRD2) antagonist Raclopride (RAC). Neuropeptide correlations observed in MR were non-existent in NR monkeys. Compared to MR, NR showed reduced DA tone; higher basal serum T; and lower CSF serotonin (5-HT). RAC increased PRL, T and CORT, but the magnitude of responses varied as a function of rearing. Levels of PRL significantly increased following RAC in MR, but not NR. Elevations in T following RAC were only significant among MR. Contrastingly, the net change (RAC CORT – saline CORT) in CORT was greater in NR than MR. Finally, observations conducted during the juvenile phase in a novel play-arena revealed more aggressive, self-injurious, and repetitive behaviors, which negatively correlated with indexes of dopaminergic tone in NR monkeys. In conclusion, early maternal deprivation alters brain DA systems, and thus may be associated with characteristic cognitive, social, and addiction outcomes.

Validation of the Social Responsiveness Scale (SRS) to screen for atypical social behaviors in juvenile macaques.

Primates form strong social bonds and depend on social relationships and networks that provide shared resources and protection critical for survival. Social deficits such as those present in autism spectrum disorder (ASD) and other psychiatric disorders hinder the individual’s functioning in communities. Given that early diagnosis and intervention can improve outcomes and trajectories of ASD, there is a great need for tools to identify early markers for screening/diagnosis, and for translational animal models to uncover biological mechanisms and develop treatments. One of the most widely used screening tools for ASD in children is the Social Responsiveness Scale (SRS), a quantitative measure used to identify individuals with atypical social behaviors. The SRS has been adapted for use in adult rhesus monkeys (Macaca mulatta)–a species very close to humans in terms of social behavior, brain anatomy/connectivity and development–but has not yet been validated or adapted for a necessary downward extension to younger ages matching those for ASD diagnosis in children. The goal of the present study was to adapt and validate the adult macaque SRS (mSRS) in juvenile macaques with age equivalent to mid-childhood in humans. Expert primate coders modified the mSRS to adapt it to rate atypical social behaviors in juvenile macaques living in complex social groups at the Yerkes National Primate Research Center. Construct and face validity of this juvenile mSRS (jmSRS) was determined based on well-established and operationalized measures of social and non-social behaviors in this species using traditional behavioral observations. We found that the jmSRS identifies variability in social responsiveness of juvenile rhesus monkeys and shows strong construct/predictive validity, as well as sensitivity to detect atypical social behaviors in young male and female macaques across social status. Thus, the jmSRS provides a promising tool for translational research on macaque models of children social disorders.

Gonadotrophin-mediated miRNA expression in testis at onset of puberty in rhesus monkey: predictions on regulation of thyroid hormone activity and DLK1-DIO3 locus.

Molecular mechanisms responsible for the initiation of primate spermatogenesis remain poorly characterized. Previously, 48 h stimulation of the testes of three juvenile rhesus monkeys with pulsatile LH and FSH resulted in down-regulation of a cohort of genes recognized to favor spermatogonia stem cell renewal. This change in genetic landscape occurred in concert with amplification of Sertoli cell proliferation and the commitment of undifferentiated spermatogonia to differentiate. In this report, the non-protein coding small RNA transcriptomes of the same testes were characterized using RNA sequencing: 537 mature micro-RNAs (miRNAs), 322 small nucleolar RNAs (snoRNAs) and 49 small nuclear RNAs (snRNAs) were identified. Pathway analysis of the 20 most highly expressed miRNAs suggested that these transcripts contribute to limiting the proliferation of the primate Sertoli cell during juvenile development. Gonadotrophin treatment resulted in differential expression of 35 miRNAs, 12 snoRNAs and four snRNA transcripts. Ten differentially expressed miRNAs were derived from the imprinted delta-like homolog 1-iodothyronine deiodinase 3 (DLK1-DIO3) locus that is linked to stem cell fate decisions. Four gonadotrophin-regulated expressed miRNAs were predicted to trigger a local increase in thyroid hormone activity within the juvenile testis. The latter finding leads us to predict that, in primates, a gonadotrophin-induced selective increase in testicular thyroid hormone activity, together with the established increase in androgen levels, at the onset of puberty is necessary for the normal timing of Sertoli cell maturation, and therefore initiation of spermatogenesis. Further examination of this hypothesis requires that peripubertal changes in thyroid hormone activity of the testis of a representative higher primate be determined empirically.

Fluoxetine Administration in Juvenile Monkeys: Implications for Pharmacotherapy in Children.

Fluoxetine therapy has been approved for children with major depressive disorder and obsessive compulsive disorder for over 14 years and has expanded to other childhood behavior disorders. As use increases, more detail on fluoxetine effects during juvenile brain development can help maintain safe and effective use of this therapy. Here, a narrative review is provided of previously published findings from a large nonhuman primate project. Fluoxetine was administered to juvenile male rhesus monkeys for an extended period (2 years) prior to puberty. Compared to controls, treated monkeys showed sleep disruption, facilitated social interaction, greater impulsivity, and impaired sustained attention during treatment. No effects on growth were seen. Metabolomics assays characterized a distinctive response to fluoxetine and demonstrated individual differences that were related to the impulsivity measure. Fluoxetine interactions with monoamine oxidase A polymorphisms that influenced behavior and metabolomics markers were an important, previously unrecognized finding of our studies. After treatment was discontinued, some behavioral effects persisted, but short-term memory and cognitive flexibility testing did not show drug effects. This detailed experimental work can contribute to clinical research and continued safe and effective fluoxetine pharmacotherapy in children.

Does behavioral flexibility contribute to successful play among juvenile rhesus macaques?

Animal play often resembles aggressive interactions, making it difficult for players and third parties to distinguish between the two types of behavior or to concur on aspects of play. In this sense, social play involves some degree of social risk, and players may benefit by behaving flexibly particularly when playing with unmatched partners. Here, we ask (1) whether social play among free-ranging juvenile rhesus monkeys (Macaca mulatta) is more likely to fail when partners are unmatched by sex, age, rank/kinship, or when their mothers are nearby and (2) whether juveniles behave flexibly to overcome these social risks. We first identify the factors contributing to play failure, by describing social attributes that are associated with negative outcomes of play. We then compare behavior for matched vs. unmatched partners by examining tendencies to (1) refrain from play, (2) engage in short play durations, and (3) use enhanced play signaling. Males were responsive to several play failure factors; they disproportionately used enhanced play signaling and played for short durations with unmatched partners, suggesting that they have social knowledge that supports attempts to cope flexibly with diverse play partners/situations. Females were less actively responsive to these factors. Although they refrained from playing with many unmatched partners, they did not adjust play tactics to the same degree. These sex differences may be related to differences in life histories; males preparing to disperse eventually may benefit from expanding their social networks through play, while philopatric females may have less need to do so. While social play provides many benefits for animals, play attempts may also involve risks of failure, from refusal by partners to escalation into aggression, particularly when players are mismatched physically or socially. Growing juveniles in despotic societies may be especially vulnerable to such risk. We ask whether juvenile rhesus monkeys behave flexibly when playing with mismatched partners in a way that may help them overcome such risks. We demonstrate that males, who typically emigrate from their natal groups, are indeed sensitive to mismatches in social characteristics or situations; they play in short durations and enhance their play signaling during play sessions involving mismatches. Females, who permanently remain in their maternal groups are less responsive. These sex differences suggest that juvenile males may hone social skills via playful interaction in preparation for emigration, while females have less need to do so.

The testicular transcriptome associated with spermatogonia differentiation initiated by gonadotrophin stimulation in the juvenile rhesus monkey (Macaca mulatta).

What is the genetic landscape within the testis of the juvenile rhesus monkey (Macaca mulatta) that underlies the decision of undifferentiated spermatogonia to commit to a pathway of differentiation when puberty is induced prematurely by exogenous LH and FSH stimulation? Forty-eight hours of gonadotrophin stimulation of the juvenile monkey testis resulted in the appearance of differentiating B spermatogonia and the emergence of 1362 up-regulated and 225 down-regulated testicular mRNAs encoding a complex network of proteins ranging from enzymes regulating Leydig cell steroidogenesis to membrane receptors, and from juxtacrine and paracrine factors to transcriptional factors governing spermatogonial stem cell fate. Our understanding of the cell and molecular biology underlying the fate of undifferentiated spermatogonia is based largely on studies of rodents, particularly of mice, but in the case of primates very little is known. The present study represents the first attempt to comprehensively address this question in a highly evolved primate. Global gene expression in the testis from juvenile rhesus monkeys that had been stimulated with recombinant monkey LH and FSH for 48 h (N = 3) or 96 h (N = 4) was compared to that from vehicle treated animals (N = 3). Testicular cell types and testosterone secretion were also monitored. Precocious testicular puberty was initiated in juvenile rhesus monkeys, 14-24 months of age, using a physiologic mode of intermittent stimulation with i.v. recombinant monkey LH and FSH that within 48 h produced 'adult' levels of circulating LH, FSH and testosterone. Mitotic activity was monitored by immunohistochemical assays of 5-bromo-2'-deoxyuridine and 5-ethynyl-2'-deoxyuridine incorporation. Animals were bilaterally castrated and RNA was extracted from the right testis. Global gene expression was determined using RNA-Seq. Differentially expressed genes (DEGs) were identified and evaluated by pathway analysis. mRNAs of particular interest were also quantitated using quantitative RT-PCR. Fractions of the left testis were used for histochemistry or immunoflouresence. Differentiating type B spematogonia were observed after both 48 and 96 h of gonadotrophin stimulation. Pathway analysis identified five super categories of over-represented DEGs. Repression of GFRA1 (glial cell line-derived neurotrophic factor family receptor alpha 1) and NANOS2 (nanos C2HC-type zinc finger 2) that favor spermatogonial stem cell renewal was noted after 48 and 96 h of LH and FSH stimulation. Additionally, changes in expression of numerous genes involved in regulating the Notch pathway, cell adhesion, structural plasticity and modulating the immune system were observed. Induction of genes associated with the differentiation of spermatogonia stem cells (SOHLH1(spermatogenesis- and oogenesis-specific basic helix-loop-helix 1), SOHLH2 and KIT (V-Kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog)) was not observed. Expression of the gene encoding STRA8 (stimulated by retinoic acid 8), a protein generally considered to mark activation of retinoic acid signaling, was below our limit of detection. The entire mRNA data set for vehicle and gonadotrophin treated animals (N = 10) has been deposited in the GEO-NCBI repository (GSE97786). The limited number of monkeys per group and the dilution of low abundance germ cell transcripts by mRNAs contributed from somatic cells likely resulted in an underestimation of the number of differentially expressed germ cell genes. The findings that expression of GDNF (a major promoter of spermatogonial stem cell renewal) was not detected in the control juvenile testes, expression of SOHLH1, SOHLH2 and KIT, promoters of spermatogonial differentiation in mice, were not up-regulated in association with the gonadotrophin-induced generation of differentiating spermatogonia, and that robust activation of the retinoic acid signaling pathway was not observed, could not have been predicted. These unexpected results underline the importance of non-human primate models in translating data derived from animal research to the human situation. The work described was funded by NIH grant R01 HD072189 to T.M.P. P.A. was supported by an Endocrine Society Summer Research Fellowship Award and CONICET (Argentine Research Council), S.N. by a grant from Vali-e-Asr Reproductive Health Research Center of Tehran University of Medical Sciences (grant #24335-39-92) to Dr Batool Hosseini Rashidi, and M.P.H. by grants from the National Health and Medical Research Council of Australia, and the Victorian State Government's Operational Infrastructure Support Program. The authors have nothing to disclose.

The Helicobacter pylori type IV secretion system promotes IL-8 synthesis in a model of pediatric airway epithelium via p38 MAP kinase.

Epidemiologic studies have reported an inverse relationship between childhood Helicobacter pylori infection and development of allergic asthma. Because lung epithelium plays an important role in allergic asthma pathogenesis, we hypothesized that H. pylori may directly influence airway epithelial cell innate immune function, particularly in early childhood. To test our hypothesis, we established an in vitro H. pylori infection model using primary tracheobronchial epithelial cell cultures derived from infant, juvenile and adult rhesus monkeys. Airway epithelial cell cultures were infected with wild-type or cag pathogenicity island mutant H. pylori strains, followed by evaluation of IL-8 and IL-6 protein synthesis. We found that H. pylori primarily increased IL-8 synthesis in a MOI and age-dependent fashion, with a greater than 4-fold induction in infant versus adult cultures. H. pylori-induced IL-8 synthesis in infant and juvenile cultures was significantly reduced by cag pathogenicity island mutants, indicating a requirement for the type IV secretion system. Although peptidoglycan recognition of nucleotide binding oligomerization domain-containing protein 1 (NOD1) and NF-kappaB have been implicated as key cytokine signaling molecules for H. pylori infection in gastric epithelium, NOD1 (ML130) or NF-kappaB (JSH-23) inhibitors minimally affected IL-8 synthesis in airway epithelial cell cultures following H. pylori infection. In contrast, inhibition of the p38 MAP kinase pathway (SB203580) resulted in almost complete suppression of H. pylori-induced IL-8 synthesis. Collectively, these results indicate that H. pylori can preferentially elicit IL-8 synthesis in a model of pediatric airway epithelium using the type IV secretion system via p38 MAP kinase.

Cognitive performance of juvenile monkeys after chronic fluoxetine treatment

Potential long term effects on brain development are a concern when drugs are used to treat depression and anxiety in childhood. In this study, male juvenile rhesus monkeys (three-four years of age) were dosed with fluoxetine or vehicle (N = 16/group) for two years. Histomorphometric examination of cortical dendritic spines conducted after euthanasia at one year postdosing (N = 8/group) suggested a trend toward greater dendritic spine synapse density in prefrontal cortex of the fluoxetine-treated monkeys. During dosing, subjects were trained for automated cognitive testing, and evaluated with a test of sustained attention. After dosing was discontinued, sustained attention, recognition memory and cognitive flexibility were evaluated. Sustained attention was affected by fluoxetine, both during and after dosing, as indexed by omission errors. Response accuracy was not affected by fluoxetine in post-dosing recognition memory and cognitive flexibility tests, but formerly fluoxetine-treated monkeys compared to vehicle controls had more missed trial initiations and choices during testing. Drug treatment also interacted with genetic and environmental variables: MAOA genotype (high- and low transcription rate polymorphisms) and testing location (upper or lower tier of cages). Altered development of top-down cortical regulation of effortful attention may be relevant to this pattern of cognitive test performance after juvenile fluoxetine treatment.

Longitudinal Evaluation of Eye Misalignment and Eye Movements Following Surgical Correction of Strabismus in Monkeys.

PurposeStrabismus correction surgery is well documented in both the literature and practice with varying levels of success and permanence. Our goal was to characterize longitudinal changes in eye alignment and eye movements following strabismus correction surgery in a monkey model for developmental strabismus.MethodsWe studied two juvenile rhesus monkeys with exotropia previously induced via an optical prism-rearing paradigm in infancy. Eye misalignment was corrected via a resection–recession surgery of the horizontal rectus muscles of one eye. Binocular search coils were used to collect eye movement data during smooth-pursuit, saccades, and fixation tasks before surgical treatment, immediately after surgery, and through 6 months after treatment.ResultsBoth animals showed an immediate ∼70% reduction in misalignment as a consequence of surgery that regressed to a 20%–40% improvement by 6 months after treatment. Significant changes were observed in saccade and smooth-pursuit gain of the nonviewing eye after surgery, which also reverted to presurgical values by 6 months. A temporary improvement in fixation stability of the nonviewing eye was observed after surgery; naso-temporal (N/T) asymmetry of monocular smooth-pursuit remained unchanged.ConclusionsSurgical realignment is followed by plastic changes that often lead to reversal of surgery effects. Immediate improvement in misalignment and changes in eye movement gains are likely a result of contractility changes at the level of the extraocular muscle, whereas longer-term effects are likely a combination of neural and muscle adaptation.

Peripheral fibroblast metabolic pathway alterations in juvenile rhesus monkeys undergoing long-term fluoxetine administration

We report on biochemical pathways perturbed upon chronic fluoxetine administration to juvenile macaques using global metabolomics analyses of fibroblasts derived from skin biopsies. After exposure to tissue culture conditions confounding environmental factors are eliminated and identification of metabolites whose levels are affected by the drug become apparent with a better signal-to-noise ratio compared to data obtained from plasma and cerebrospinal fluid (CSF). Levels of more than 200 metabolites were analyzed to interrogate affected molecular pathways and identify biomarkers of drug response. In addition, we have correlated the metabolomics results with monoamine oxidase (MAOA) genotype and impulsivity behavioral data. Affected pathways include Purine and Pyrimidine metabolisms that have been previously implicated to contribute to neuropsychiatric disorders.

Sleep disturbance as detected by actigraphy in pre-pubertal juvenile monkeys receiving therapeutic doses of fluoxetine

Sleep disturbance is a reported side effect of antidepressant drugs in children. Using a nonhuman primate model of childhood selective serotonin reuptake inhibitor (SSRI) therapy, sleep was studied quantitatively with actigraphy. Two 48-h sessions were recorded in the home cage environment of juvenile male rhesus monkeys at two and three years of age, after one and two years of treatment with a therapeutic dose of the SSRI fluoxetine, and compared to vehicle treated controls. A third session was conducted one year after discontinuation of treatment at four years of age. During treatment, the fluoxetine group demonstrated sleep fragmentation as indexed by a greater number of rest–activity transitions compared to controls. In addition fluoxetine led to more inactivity during the day as indexed by longer duration of rest periods and the reduced activity during these periods. The fluoxetine effect on sleep fragmentation, but not on daytime rest, was modified by the monkey's genotype for polymorphisms of monoamine oxidase A (MAOA), an enzyme that metabolizes serotonin. After treatment, the fluoxetine effect on nighttime rest–activity transitions persisted, but daytime activity was not affected. The demonstration in this nonhuman primate model of sleep disturbance in connection with fluoxetine treatment and specific genetic polymorphisms, and in the absence of diagnosed psychopathology, can help inform use of this drug in children.

Peer social interaction is facilitated in juvenile rhesus monkeys treated with fluoxetine

Fluoxetine improves social interactions in children with autism, social anxiety and social phobia. It is not known whether this effect is mediated directly or indirectly by correcting the underlying pathology. Genetics may also influence the drug effect. Polymorphisms of the MAOA (monoamine oxidase A) gene interact with fluoxetine to influence metabolic profiles in juvenile monkeys. Juvenile nonhuman primates provide an appropriate model for studying fluoxetine effects and drug*gene interactions in children.Male rhesus monkeys 1–3 years of age living in permanent social pairs were treated daily with a therapeutic dose of fluoxetine or vehicle (n = 16/group). Both members of each social pair were assigned to the same treatment group. They were observed for social interactions with their familiar cagemate over a 2-year dosing period. Subjects were genotyped for MAOA variable number of tandem repeats (VNTR) polymorphisms categorized for high or low transcription rates (hi-MAOA, low-MAOA).Fluoxetine-treated animals spent 30% more time in social interaction than vehicle controls. Fluoxetine significantly increased the duration of quiet interactions, the most common type of interaction, and also of immature sexual behavior typical of rhesus in this age group. Specific behaviors affected depended on MAOA genotype of the animal and its social partner. When given fluoxetine, hi-MOAO monkeys had more social invitation and initiation behaviors and low-MAOA subjects with low-MAOA partners had more grooming and an increased frequency of some facial and vocal expressive behaviors.Fluoxetine may facilitate social interaction in children independent of remediation of psychopathology. Common genetic variants may modify this effect.

Bone growth in juvenile rhesus monkeys is influenced by 5HTTLPR polymorphisms and interactions between 5HTTLPR polymorphisms and fluoxetine

Male rhesus monkeys received a therapeutic oral dose of the selective serotonin reuptake inhibitor (SSRI) fluoxetine daily from 1 to 3years of age. Puberty is typically initiated between 2 and 3years of age in male rhesus and reproductive maturity is reached at 4years. The study group was genotyped for polymorphisms in the monoamine oxidase A (MAOA) and serotonin transporter (SERT) genes that affect serotonin neurotransmission. Growth was assessed with morphometrics at 4month intervals and radiographs of long bones were taken at 12month intervals to evaluate skeletal growth and maturation. No effects of fluoxetine, or MAOA or SERT genotype were found for growth during the first year of the study. Linear growth began to slow during the second year of the study and serotonin reuptake transporter (SERT) long polymorphic region (5HTTLPR) polymorphism effects with drug interactions emerged. Monkeys with two SERT 5HTTLPR L alleles (LL, putative greater transcription) had 25–39% less long bone growth, depending on the bone, than monkeys with one S and one L allele (SL). More advanced skeletal maturity was also seen in the LL group, suggesting earlier onset of puberty. An interaction between 5HTTLPR polymorphisms and fluoxetine was identified for femur and tibia growth; the 5HTTLPR effect was seen in controls (40% less growth for LL) but not in the fluoxetine treated group (10% less growth for LL). A role for serotonin in peripubertal skeletal growth and maturation has not previously been investigated but may be relevant to treatment of children with SSRIs.