We recently reported that in aged female rhesus macaques, spatial learning and memory correlates with circadian sleep-wake measures and hippocampal muscarinic type 1 (M1) receptor binding. To investigate if spatial memory also correlates with measures of immune function, we now assessed the magnitude of the adaptive immune response to vaccination in the same old female rhesus macaques. Cognitively characterized animals were classified as good spatial performers (GSP) or poor spatial performers (PSP) based on performance in the Spatial Foodport maze. The GSP group had higher frequency of CD8, but not CD4, interferon-γ (IFN-γ) producing cells following vaccination compared to the PSP group, suggesting a stronger CD8 T cell response in the GSP group. In addition, the number of CD-8 IFN-γ positive cells correlated with measures of sleep quality. Interestingly, the PSP group had a significantly higher antibody titer compared to the GSP group, and antibody titer negatively correlated with day-time activity. Thus, in aged female rhesus macaques, superior cognitive performance is correlated with a more robust CD8 T cell response but a reduced antibody response to vaccination.
spatial learning and memory; immune senescence; circadian activity
Adaptive CD4+ and CD8+ T-cell responses have been associated with control of human immunodeficiency virus/simian immunodeficiency virus (HIV/SIV) replication. Here, we have designed a study with Indian rhesus macaques to more directly assess the role of CD8 SIV-specific responses in control of viral replication. Macaques were immunized with a DNA prime-modified vaccinia virus Ankara (MVA)-SIV boost regimen under normal conditions or under conditions of antibody-induced CD4+ T-cell deficiency. Depletion of CD4+ cells was performed in the immunized macaques at the peak of SIV-specific CD4+ T-cell responses following the DNA prime dose. A group of naïve macaques was also treated with the anti-CD4 depleting antibody as a control, and an additional group of macaques immunized under normal conditions was depleted of CD8+ T cells prior to challenge exposure to SIVmac251. Analysis of the quality and quantity of vaccine-induced CD8+ T cells demonstrated that SIV-specific CD8+ T cells generated under conditions of CD4+ T-cell deficiency expressed low levels of Bcl-2 and interleukin-2 (IL-2), and plasma virus levels increased over time. Depletion of CD8+ T cells prior to challenge exposure abrogated vaccine-induced protection as previously shown. These data support the notion that adaptive CD4+ T cells are critical for the generation of effective CD8+ T-cell responses to SIV that, in turn, contribute to protection from AIDS. Importantly, they also suggest that long-term protection from disease will be afforded only by T-cell vaccines for HIV that provide a balanced induction of CD4+ and CD8+ T-cell responses and protect against early depletion of CD4+ T cells postinfection.
The serotonin system responds to the ovarian steroid steroids, estradiol (E) and progesterone (P), in women and female animal models. In macaques, ovarian steroid administration to ovariectomized (Ovx) individuals improves serotonin neural function through actions on pivotal serotonin-related genes and proteins, such as TPH2 (tryptophan hydroxylase 2), SERT (serotonin reuptake transporter) and the 5HT1A autoreceptor. In addition, ovarian steroid administration reduces gene and protein expression in the caspase-independent pathway and reduces DNA fragmentation in serotonin neurons. This study examines the hypothesis that long-term ovariectomy will lead to a loss of serotonin neurons and compromised gene expression in serotonin neurons. Female Japanese macaques were ovariectomized or tubal-ligated (n=5/group) at 3 years of age and returned to their natal troop. After 3 years, the animals were collected, administered a fenfluramine challenge to determine global serotonin availability and then euthanized. Fev, TPH2, SERT and 5HT1A expression were examined with digoxigenin in situ hybridization (dig-ISH) and quantitative image analysis. Cell number, positive pixel area and average pixel density were determined. In the Ovx group, Fev, TPH2, SERT and 5HT1A showed a significant decease in average and total cell number and positive pixel area. The reduction in Fev-positive neurons suggests that there were fewer serotonin neurons in Ovx animals compared to ovary-intact animals. Compared to ovary-intact animals, SERT also showed a decrease in positive-pixel density. The decrease in TPH2 in the Ovx animals was consistent with earlier results in 5-month Ovx animals, but it may be due to the decrease in cell number rather than a decrease in expression on an individual cell basis. The decrease in SERT and 5HT1A in long-term Ovx differed from previous studies in short-term Ovx. In summary, long-term ovarian steroid loss resulted in fewer serotonin neurons and overall lower Fev, TPH2, SERT and 5HT1A gene expression. This may be due to serotonin cell death or to a negative impact on a long-term developmental process in young female macaques.
Symptoms of anxiety and depression often occur in young women after complete hysterectomy and in older women during menopause. There are many variables that are hard to control in human population studies, but that are absent to a large extent in stable nonhuman primate troops. However, macaques exhibit depressive and anxious behaviors in response to similar situations as humans such as isolation, stress, instability or aggression. Therefore, we hypothesized that examination of behavior in ovariectomized individuals in a stable macaque troop organized along matriarchal lineages and in which individuals have social support from extended family, would reveal effects that were due to the withdrawal of ovarian steroids without many of the confounds of human society. We also tested the hypothesis that ovariectomy would elicit and increase anxious behavior in a stressful situation such as brief exposure to single caging. Japanese macaques (Macaca fuscata) were ovariectomized (Ovx) or tubal-ligated (intact controls) at 3 years of age and allowed to mature for 3 years in a stable troop of approximately 300 individuals. Behaviors were recorded in the outdoor corral in the third year followed by individual temperament tests in single cages. There was no obvious difference in anxiety-related behaviors such as scratching between Ovx and tubal-ligated animals in the corral. Nonetheless, compared to tubal-ligated animals, Ovx animals exhibited a significant decrease in (1) positive social behavior, (2) initiating dominance behavior (3) time receiving grooming, (4) locomoting, (5) mounting behavior, and in (6) consort behavior. However, Ovx females exhibited a significant increase in (1) consummatory behavior and (2) object play compared to tubal-ligated controls. In the individual temperament tests, Ovx individuals exhibited an increase in anxiety-related behaviors. There was no difference in adrenal weight/body weight suggesting that neither group was under chronic stress. These data indicate that ovarian hormones enable females to successfully navigate their social situation and may reduce anxiety in novel situations.
A comparative evaluation of the immunity stimulated with a vaccine regimen that includes simian immunodeficiency virus (SIV), interleukin 2 (IL-2), and IL-15 DNAs, recombinant modified vaccinia virus Ankara (rMVA), and inactivated SIVmac239 particles administered into the oral and nasal cavities, small intestine, and vagina was carried out in female rhesus macaques to determine the best route to induce diverse anti-SIV immunity that may be critical to protection from SIV infection and disease. All four immunizations generated mucosal SIV-specific IgA. Oral immunization was as effective as vaginal immunization in inducing SIV-specific IgA in vaginal secretions and generated greater IgA responses in rectal secretions and saliva samples compared to the other immunization routes. All four immunizations stimulated systemic T-cell responses against Gag and Env, albeit to a different extent, with oral immunization providing greater magnitude and nasal immunization providing wider functional heterogeneity. SIV-specific T cells producing gamma interferon (IFN-γ) dominated these responses. Limited levels of SIV-specific IgG antibodies were detected in plasma samples, and no SIV-specific IgG antibodies were detected in secretions. Vaccination also induced CD4+ and CD8+ T-cell responses in the rectal and vaginal mucosa with greater functional heterogeneity than in blood samples. Rectal T-cell responses were significantly greater in the orally vaccinated animals than in the other animals. The most balanced, diverse, and higher-magnitude vaginal T-cell responses were observed after intestinal vaccination. Significantly higher CD8+ granzyme B-positive T-cell responses were observed systemically after intestinal vaccination and in rectal cells after oral immunization. The majority of SIV-specific T cells that produced granzyme B did not produce cytokines. Of the immunization routes tested, oral vaccination provided the most diverse and significant response to the vaccine.
Ovarian steroid hormones influence not only seizure phenomena, but also the neuronal cell death that follows. In the present study, we applied two models of ovarian steroid loss, ovariectomy and chemically-induced ovarian failure, to evaluate kainate-induced seizure activity and the susceptibility of hippocampal neurons to seizure-induced neurodegeneration. Young adult female FVB/NJ mice were ovariectomized with (OVX+E, n=6) or without (OVX, n=8) estrogen replacement. A separate group of females received the ovotoxin, 4-vinylcyclohexene diepoxide (VCD, n=8) to deplete ovarian follicles. Mice underwent kainate-induced status epilepticus and were evaluated for seizure activity (3 hours) and delayed hippocampal neuronal injury (7 days). While there were no differences in latency or duration of severe seizures among control, OVX and VCD-treated mice, OVX+E mice exhibited seizures of a significantly longer duration. However, both VCD-induced ovarian failure and OVX led to a dramatic reduction in the extent of excitotoxic cell death, with slightly greater effects observed in VCD-treated mice. Estradiol administration to OVX mice also exerted a significant neuroprotective effect against kainate-induced cell death. These results support and extend earlier findings suggesting that the hormonal milieu may have differential effects on seizure susceptibility that are separate and distinct from those influencing hippocampal neuronal vulnerability. Collectively, these findings highlight the complex interactions among the loss of ovarian steroid hormones, estrogen replacement, seizures, and seizure-induced cell death.
Estrogen; Progesterone; 4-Vinylcyclohexene diepoxide; Ovariectomy; Neuroprotection
Induction of virus-specific T-cell responses in mucosal as well as systemic compartments of the immune system is likely to be a critical feature of an effective AIDS vaccine. We investigated whether virus-specific CD8+ lymphocytes induced in rhesus macaques by immunization with attenuated simian immunodeficiency virus (SIV), an approach that is highly effective in eliciting protection against mucosal challenge, express the mucosa-homing receptor α4β7 and traffic to the intestinal mucosa. SIV-specific CD8+ T cells expressing α4β7 were detected in peripheral blood and intestine of macaques infected with attenuated SIV. In contrast, virus-specific T cells in blood of animals immunized cutaneously by a combined DNA-modified vaccinia virus Ankara regimen did not express α4β7. These results demonstrate the selective induction of SIV-specific CD8+ T lymphocytes expressing α4β7 by a vaccine approach that replicates in mucosal tissue and suggest that induction of virus-specific lymphocytes that are able to home to mucosal sites may be an important characteristic of a successful AIDS vaccine.
Diminished estrogen influence at menopause is reported to be associated with cognitive decline, heightened anxiety and hypertension. While estrogen therapy is often prescribed to overcome these behavioral and physiological deficits, antioxidants which have been shown beneficial are gaining nutritional intervention and popularity. Therefore, in the present study, utilizing the antioxidant properties of grapes, we have examined effect of 3 weeks of grape powder (GP; 15 g/L dissolved in tap water) treatment on anxiety-like behavior, learning-memory impairment and high blood pressure in ovariectomized (OVX) rats. Four groups of female Wistar rats were used; sham control, sham-GP treated, OVX and OVX+GP treated. We observed a significant increase in systolic and diastolic blood pressure in OVX rats as compared to sham-controls. Furthermore, ovariectomy increased anxiety-like behavior and caused learning and memory impairment in rats as compared to sham-controls. Interestingly, providing grape powder treated water to OVX rats restored both systolic and diastolic blood pressure, decreased anxiety-like behavior and improved memory function. Moreover, OVX rats exhibited an impaired long term potentiation which was restored with grape powder treatment. Furthermore, ovariectomy increased oxidative stress in the brain, serum and urine, selectively decreasing antioxidant enzyme, glyoxalase-1 protein expression in the hippocampus but not in the cortex and amygdala of OVX rats, while grape powder treatment reversed these effects. Other antioxidant enzyme levels, including manganese superoxide dismutase (SOD) and Cu/Zn SOD remained unchanged. We suggest that grape powder by regulating oxidative stress mechanisms exerts its protective effect on blood pressure, learning-memory and anxiety-like behavior. Our study is the first to examine behavioral, biochemical, physiological and electrophysiological outcome of estrogen depletion in rats and to test protective role of grape powder, all in the same study.
Aged individuals are more susceptible to infections due to a general decline in immune function broadly referred to as immune senescence. While age-related changes in the adaptive immune system are well documented, aging of the innate immune system remains less well understood, particularly in nonhuman primates. A more robust understanding of age-related changes in innate immune function would provide mechanistic insight into the increased susceptibility of the elderly to infection. Rhesus macaques have proved a critical translational model for aging research, and present a unique opportunity to dissect age-dependent modulation of the innate immune system. We examined age-related changes in: (i) innate immune cell frequencies; (ii) expression of pattern recognition receptors (PRRs) and innate signaling molecules; (iii) cytokine responses of monocytes and dendritic cells (DC) following stimulation with PRR agonists; and (iv) plasma cytokine levels in this model. We found marked changes in both the phenotype and function of innate immune cells. This included an age-associated increased frequency of myeloid DC (mDC). Moreover, we found toll-like receptor (TLR) agonists lipopolysaccharide (TLR4), fibroblast stimulating ligand-1 (TLR2/6), and ODN2006 (TLR7/9) induced reduced cytokine responses in aged mDC. Interestingly, with the exception of the monocyte-derived TNFα response to LPS, which increased with age, TNFα, IL-6, and IFNα responses declined with age. We also found that TLR4, TLR5, and innate negative regulator, sterile alpha and TIR motif containing protein (SARM), were all expressed at lower levels in young animals. By contrast, absent in melanoma 2 and retinoic acid-inducible gene I expression was lowest in aged animals. Together, these observations indicate that several parameters of innate immunity are significantly modulated by age and contribute to differential immune function in aged macaques.
nonhuman primate; innate immunity; myeloid; immune senscence; pattern recognition receptor
Poxvirus vectors have proven to be highly effective for boosting immune responses in diverse vaccine settings. Recent reports reveal marked differences in the gene expression of human dendritic cells infected with two leading poxvirus-based human immunodeficiency virus (HIV) vaccine candidates, New York vaccinia virus (NYVAC) and modified vaccinia virus Ankara (MVA). To understand how complex genomic changes in these two vaccine vectors translate into antigen-specific systemic immune responses, we undertook a head-to-head vaccine immunogenicity and efficacy study in the pathogenic HIV type 1 (HIV-1) model of AIDS in Indian rhesus macaques. Differences in the immune responses in outbred animals were not distinguished by enzyme-linked immunospot assays, but differences were distinguished by multiparameter fluorescence-activated cell sorter analysis, revealing a difference between the number of animals with both CD4+ and CD8+ T-cell responses to vaccine inserts (MVA) and those that elicit a dominant CD4+ T-cell response (NYVAC). Remarkably, vector-induced differences in CD4+/CD8+ T-cell immune responses persisted for more than a year after challenge and even accompanied antigenic modulation throughout the control of chronic infection. Importantly, strong preexposure HIV-1/simian immunodeficiency virus-specific CD4+ T-cell responses did not prove deleterious with respect to accelerated disease progression. In contrast, in this setting, animals with strong vaccine-induced polyfunctional CD4+ T-cell responses showed efficacies similar to those with stronger CD8+ T-cell responses.
The neuroprotective effects of estrogen in young adult rodents are well established. Less well understood is how estrogen neuroprotection is affected by aging and interactions with progesterone. In this study, we investigated the effects of estrogen and continuous progesterone, both alone and in combination, on hippocampal neuron survival following kainate lesion in 14 month-old female rats entering reproductive senescence. Our results show that ovariectomy-induced hormone depletion did not significantly affect the extent of kainate-induced neuron loss. Treatment of ovariectomized rats with estrogen significantly reduced neuron loss, however this effect was blocked by co-administration of continuous progesterone. Treatment of ovariectomized rats with progesterone alone did not significantly affect kainate toxicity. These results provide new insight into factors that regulate estrogen neuroprotection, which has important implications for hormone therapy in postmenopausal women.
estrogen; progesterone; neuroprotection; kainite; Alzheimer’s disease
Human immunodeficiency virus (HIV)/simian immunodeficiency virus (SIV) infection causes B-cell dysregulation and the loss of memory B cells in peripheral blood mononuclear cells (PBMC). These effects are not completely reversed by antiretroviral treatment (ART). To further elucidate B-cell changes during chronic SIV infection and treatment, we investigated memory B-cell subpopulations and plasma cells/plasmablasts (PC/PB) in blood, bone marrow, and lymph nodes of rhesus macaques during ART and upon release from ART. Macaques previously immunized with SIV recombinants and the gp120 protein were included to assess the effects of prior vaccination. ART was administered for 11 weeks, with or without gp120 boosting at week 9. Naïve and resting, activated, and tissue-like memory B cells and PC/PB were evaluated by flow cytometry. Antibody-secreting cells (ASC) and serum antibody titers were assessed. No lasting changes in B-cell memory subpopulations occurred in bone marrow and lymph nodes, but significant decreases in numbers of activated memory B cells and increases in numbers of tissue-like memory B cells persisted in PBMC. Macaque PC/PB were found to be either CD27+ or CD27− and therefore were defined as CD19+ CD38hi CD138+. The numbers of these PC/PB were transiently increased in both PBMC and bone marrow following gp120 boosting of the unvaccinated and vaccinated macaque groups. Similarly, ASC numbers in PBMC and bone marrow of the two macaque groups also transiently increased following envelope boosting. Nevertheless, serum binding titers against SIVgp120 remained unchanged. Thus, even during chronic SIV infection, B cells respond to antigen, but long-term memory does not develop, perhaps due to germinal center destruction. Earlier and/or prolonged treatment to allow the generation of virus-specific long-term memory B cells should benefit ART/therapeutic vaccination regimens.
Rhesus macaque models have been instrumental for the development and testing of vaccines prior to human studies and have provided fundamental insights into the determinants of immune efficacy in a variety of infectious diseases. However, the characterization of antigen-specific T cell receptor (TCR) repertoires during adaptive immune responses in these models has previously relied on human TCR gene assignments. Here, we extracted and characterized TCR β-chain (TRB) genes from the recently sequenced rhesus macaque genome that are homologous to the human TRB genes. Comparison of the rhesus macaque TRB genes with the human TRB genes revealed an average best-match similarity of 92.9%. Furthermore, we confirmed the usage of most rhesus macaque TRB genes by expressed TCRβ sequences within epitope-specific TCR repertoires. This primary description of the rhesus macaque TRB genes will provide a standardized nomenclature and enable better characterization of TCR usage in studies that utilize this species.
T cell; T cell receptor; T cell receptor repertoire
Linear dominance hierarchies organize and maintain stability in female rhesus macaque (Macaca mulatta) social groups regardless of group size. As a consequence of their low social status, subordinate females suffer from an array of adverse outcomes including reproductive compromise, impaired immune function, and poor cardiovascular health. However, data that differentiate limbic-hypothalamic-pituitary adrenal axis (LHPA) parameters between dominant from subordinate female monkeys are inconsistent, bringing into question whether social subordination alters the LHPA axis in female macaques. One difficulty in examining LHPA function in macaques may be the confounding effects of cycling ovarian steroids that are known to modulate LHPA activity. The current study used ovariectomized dominant and subordinate female rhesus monkeys to examine the effect that social subordination has on LHPA function by measuring morning and diurnal serum cortisol levels, dexamethasone (Dex) suppression of cortisol, metabolic clearance of Dex, and ACTH stimulation of adrenal cortisol release and cortisol response following exposure to acute social isolation. Compared to dominant females, subordinate females showed diminished morning peak cortisol secretion, weakened glucocorticoid negative feedback, and decreased adrenal cortisol response to an ACTH challenge as well as a restrained cortisol response following social isolation. However, the metabolism of Dex did not account for differences in Dex suppression between dominant and subordinate females. These results indicate that the ability to mount and limit glucocorticoid release is significantly reduced by psychosocial stress in female rhesus macaques, suggesting a hyporesponsive LHPA phenotype which resembles that observed in several human psychopathologies.
social subordination; dexamethasone; cortisol; ACTH; psychosocial stress; monkeys
The molecular mechanisms for the discrepancy in outcome of initiating estrogen therapy (ET) around peri-menopause or several years after menopause in women are unknown. We hypothesize that the level of expression of a dominant negative estrogen receptor (ER) β variant, ERβ2, may be a key factor determining the effectiveness of ET in post-menopausal women. We tested this hypothesis in ovariectomized nine month-old (an age when irregular estrous cycles occur) female Sprague Dawley rats. Estradiol treatment was initiated either 6 days (Early ET, analogous to 4 months post-menopause in humans), or 180 days (Late ET, analogous to 11 years post-menopause in humans) after ovariectomy. Although ERβ2 expression increased in all OVX rats, neurogenic and neuroprotective responses to estradiol differed in Early and Late ET. Early ET reduced ERβ2 expression in both hippocampus and white blood cells, increased the hippocampal cell proliferation as assessed by Ki-67 expression, and improved mobility in the forced swim test. Late ET resulted in either no or modest effects on these parameters. There was a close correlation between the degree of ERβ2 expression and the preservation of neural effects by ET after OVX in rats, supporting the hypothesis that persistent elevated levels of ERβ2 are a molecular basis for the diminished effectiveness of ET in late post-menopausal women. The correlation between the expression of ERβ2 in circulating white blood cells and brain cells suggests that ERβ2 expression in peripheral blood cells may be an easily accessible marker to predict the effective window for ET in the brain.
Immune senescence, defined as the age-associated dysregulation and dysfunction of the immune system, is characterised by impaired protective immunity and decreased efficacy of vaccines. Recent clinical, epidemiological and immunological studies suggest that Cytomegalovirus (CMV) infection may be associated with accelerated immune senescence, possibly by restricting the naïve T cell repertoire. However, direct evidence whether and how CMV-infection is implicated in immune senescence is still lacking. In this study, we have investigated whether latent mouse CMV (MCMV) infection with or without thymectomy (Tx) alters antiviral immunity of young and aged mice. After infection with lymphocytic choriomeningitis virus (LCMV) or Vaccinia virus, specific antiviral T cell responses were significantly reduced in old, old MCMV-infected and/or Tx mice compared to young mice. Importantly, control of LCMV replication was more profoundly impaired in aged MCMV-infected mice compared to age-matched MCMV-naïve or young mice. In addition, latent MCMV infection was associated with slightly reduced vaccination efficacy in old Tx mice. In contrast to the prevailing hypothesis of a CMV-mediated restriction of the naïve T cell repertoire, we found similar naïve T cell numbers in MCMV-infected and non-infected mice, whereas ageing and Tx clearly reduced the naïve T cell pool. Instead, MCMV-infection expanded the total CD8+ T cell pool by a massive accumulation of effector memory T cells. Based on these results, we propose a new model of increased competition between CMV-specific memory T cells and any ‘de novo’ immune response in aged individuals. In summary, our results directly demonstrate in a mouse model that latent CMV-infection impairs immunity in old age and propagates immune senescence.
Cytomegalovirus (CMV) persistently infects 50–90% of the human population. After primary infection, constant immune surveillance is required to prevent CMV-related disease. During ageing, increasing T cell resources are expended to keep CMV under control. Recent human studies have suggested that this investment may come at the cost of accelerated immune senescence, a condition describing the age-associated decline of the immune system's functionality. In the present study, we have developed a mouse model to directly investigate whether and how CMV-infection might impair immunity of aged individuals. We demonstrate that old mice with long-lasting CMV-infection are more susceptible to viral infections than old mice without CMV since their virus specific T cell response is suppressed. Contrary to the prevailing hypothesis we found no indication for a CMV-associated shrinking of the naïve T cell compartment. Instead, CMV-infection precipitated a massive expansion of memory T cells. Thus, we propose an alternative mechanism of CMV-enhanced immune senescence based on T cell competition between CMV-specific memory T cells and de novo generated T cell responses. In summary, we provide the first direct evidence that CMV-infection is indeed a propagating factor for poor immunity in the elderly.
The immunogenicity and protective efficacy of a modified vaccinia virus Ankara (MVA) recombinant expressing the simian immunodeficiency virus (SIV) Gag-Pol proteins (MVA-gag-pol) was explored in rhesus monkeys expressing the major histocompatibility complex (MHC) class I allele, MamuA*01. Macaques received four sequential intramuscular immunizations with the MVA-gag-pol recombinant virus or nonrecombinant MVA as a control. Gag-specific cytotoxic T-lymphocyte (CTL) responses were detected in all MVA-gag-pol-immunized macaques by both functional assays and flow cytometric analyses of CD8+ T cells that bound a specific MHC complex class I-peptide tetramer, with levels peaking after the second immunization. Following challenge with uncloned SIVsmE660, all macaques became infected; however, viral load set points were lower in MVA-gag-pol-immunized macaques than in the MVA-immunized control macaques. MVA-gag-pol-immunized macaques exhibited a rapid and substantial anamnestic CTL response specific for the p11C, C-M Gag epitope. The level at which CTL stabilized after resolution of primary viremia correlated inversely with plasma viral load set point (P = 0.03). Most importantly, the magnitude of reduction in viremia in the vaccinees was predicted by the magnitude of the vaccine-elicited CTL response prior to SIV challenge.
Naïve Indian rhesus macaques were immunized with a mixture of optimized plasmid DNAs expressing several SIV antigens using in vivo electroporation via the intramuscular route. The animals were monitored for the development of SIV-specific systemic (blood) and mucosal (bronchoalveolar lavage) cellular and humoral immune responses. The immune responses were of great magnitude, broad (Gag, Pol, Nef, Tat, Vif), long-lasting (up to 90 weeks post 3rd vaccination) and were boosted with each subsequent immunization, even after an extended 90 week rest period. The SIV-specific cellular immune responses were consistently more abundant in BAL than in blood, and were characterized as predominantly effector memory CD4+ and CD8+ T cells in BAL and as both central and effector memory T cells in blood. SIV-specific T cells containing Granzyme B were readily detected in both blood and BAL, suggesting the presence of effector cells with cytolytic potential. DNA vaccination also elicited long-lasting systemic and mucosal humoral immune responses, including the induction of Gag-specific IgA. The combination of optimized DNA vectors and improved intramuscular delivery by in vivo electroporation has the potential to elicit both cellular and humoral responses and dissemination to the periphery, and thus to improve DNA immunization efficacy.
DNA vaccine; AIDS vaccine; rhesus macaque; BAL; PBMC; rectal mucosa; humoral immune response; cellular immune response; IFN-γ; TNFα; IL-12; adjuvant; immunology
In the rat, the sexual dimorphism in growth hormone release is driven by sex steroids, and is suggested to result mainly from differences in somatostatin (SOM) release patterns from the median eminence. We studied the effect of gonadal steroids on SOM peptide-containing cells in the periventricular nucleus (PeVN) of ovariectomized (OVX) female rats, and compared these data with data from intact male rats. Adult female rats were treated with estradiol (E2) and/or progesterone (P), 3 months (long-term) or 2 weeks (short-term) after ovariectomy (OVX). Perfusion-fixed brains were sliced and stained, and the number of SOM-immunoreactive (-ir) cells and total SOM-ir area (in μm2) were determined using computer assisted analysis. SOM-ir cells in the PeVN showed a very characteristic rostro-caudal distribution and localization in relation to the third ventricle. Both the number of SOM-ir cells and total SOM-ir area in the PeVN were higher in male compared to OVX female rats. Neither the number of SOM-ir cells, nor the total SOM-ir area in the PeVN was affected by E2 or P treatment alone. Treatment with both gonadal steroids, however, did increase total SOM-immunoreactivity. This study is the first to describe SOM cell distribution within the rat PeVN in great detail. A clear sex difference exists in SOM peptide content in the rat PeVN. In addition, E2 and P may act synergistically to affect SOM cells in the female PeVN, suggesting that both gonadal steroids may be involved in the generation of the typical feminine SOM release pattern.
Somatostatin peptide; Gonadal steroids; Growth hormone; Hypothalamus; Estrogen sensitivity; Sex difference
Sex differences have been identified in many of the behavioral and physiological effects of cannabinoids. While estrogens has been linked to some of these variations, the effects of estrogen on cannabinoid receptor binding have not been characterized within regions of the brain specifically implicated in stress responsivity and emotional behavior. To examine sex differences, and the role of estradiol, in regulation of the cannabinoid receptor, we compared the binding site density of the cannabinoid receptor within the amygdala, hippocampus and hypothalamus in males, cycling females, ovariectomized (OVX) females and estradiol-treated OVX females (OVX + E). Our data reveal that males and OVX females have higher amounts of hypothalamic and lower amounts of amygdalar cannabinoid receptor binding relative to both cycling females and OVX + E females. Within the hippocampus, ovariectomy resulted in an upregulation of cannabinoid receptor binding. These data provide a putative biochemical mechanism mediating the observed behavioral and physiological sex differences in the effects of cannabinoids, particularly with respect to stress and emotional behavior.
Ovarian hormone decline after menopause may influence cognitive performance and increase the risk for Alzheimer’s disease (AD) in women. Amyloid-β peptide (Aβ) has been proposed to be the primary cause of AD. In this study, we examined whether ovariectomy (OVX) could affect the levels of cofactors Aβ-binding alcohol dehydrogenase (ABAD) and receptor for advanced glycation endproducts (RAGE), which have been reported to potentiate Aβ-mediated neuronal perturbation, in mouse hippocampus, correlating with estrogen and Aβ levels. Female ICR mice were randomly divided into ovariectomized or sham-operated groups, and biochemical analyses were carried out at 5 weeks after the operation. OVX for 5 weeks significantly decreased hippocampal 17β-estradiol level, while it tended to reduce the hormone level in serum, compared with the sham-operated control. In contrast, OVX did not affect hippocampal Aβ1-40 level, although it significantly increased serum Aβ1-40 level. Furthermore, we demonstrated that OVX increased hippocampal ABAD level in neurons, but not astrocytes, while it did not affect RAGE level. These findings suggest that the expression of neuronal ABAD depends on estrogen level in the hippocampus and the increase in serum Aβ and hippocampal ABAD induced by ovarian hormone decline may be associated with pre-stage of memory deficit in postmenopausal women and Aβ-mediated AD pathology.
Ovariectomy; Alzheimer’s disease; β-Amyloid; ABAD
Osteoporosis, a reduction in bone mineral density, represents the most common metabolic bone disease. Postmenopausal women are particularly susceptible to osteoporosis when their production of estrogen declines. For these women, fracture is a leading cause of morbidity and mortality. This study was conducted to evaluate the protective effects of olive oil supplementation against osteoporosis in ovariectomized (OVX) rats.
We studied adult female Wistar rats aged 12-14 months, divided into three groups: sham-operated control (SHAM), ovariectomized (OVX), and ovariectomized rats supplemented with extravirgin olive oil (Olive-OVX) orally for 12 weeks; 4 weeks before ovariectomy and 8 weeks after. At the end of the experiment, blood samples were collected. Plasma levels of calcium, phosphorus, alkaline phosphatase (ALP), malondialdehyde (MDA), and nitrates were assayed. Specimens from both the tibia and the liver were processed for light microscopic examination. Histomorphometric analysis of the tibia was also performed.
The OVX-rats showed a significant decrease in plasma calcium levels, and a significant increase in plasma ALP, MDA, and nitrates levels. These changes were attenuated by olive oil supplementation in the Olive-OVX rats. Light microscopic examination of the tibia of the OVX rats revealed a significant decrease in the cortical bone thickness (CBT) and the trabecular bone thickness (TBT). In addition, there was a significant increase in the osteoclast number denoting bone resorption. In the Olive-OVX rats these parameters were markedly improved as compared to the OVX group. Examination of the liver specimens revealed mononuclear cellular infiltration in the portal areas in the OVX-rats which was not detected in the Olive-OVX rats.
Olive oil effectively mitigated ovariectomy-induced osteoporosis in rats, and is a promising candidate for the treatment of postmenopausal osteoporosis.
Previous studies demonstrated that immunization of macaques with simian immunodeficiency virus (SIV) Gag-Pol and Env recombinants of the attenuated poxvirus modified vaccinia virus Ankara (MVA) provided protection from high viremia and AIDS following challenge with a pathogenic strain of SIV. Although all animals became infected, plasma viremia was significantly reduced in animals that received the MVA-SIV recombinant vaccines compared with animals that received nonrecombinant MVA. Most importantly, the reduction in viremia resulted in a significant increase in median and cumulative survival. Continued analysis of these animals over the subsequent 9 years has shown that they maintain a survival advantage, although all but two of the macaques have progressed to AIDS. Importantly, improved survival correlated with preservation of memory CD4+ T cells in the peripheral blood. The greatest survival advantage was observed in macaques immunized with regimens containing SIV Env, and the titer of neutralizing antibodies to the challenge virus prior to or shortly following challenge correlated with preservation of CD4+ T cells. These data are consistent with a role for neutralizing antibodies in nonsterilizing protection from high viremia and associated memory CD4+ T-cell loss.
CD8α exodomain protein, a crucial immune-system factor in rhesus macaque (M. mulatta), one of the best animal models for vaccine design, was assembled and crystallized. The full structure data will contribute to future studies of immune responses in rhesus macaques.
As a T-cell co-receptor, CD8 binds to MHC class I molecules and plays a pivotal role in the activation of cytotoxic T lymphocytes. To date, structures of CD8 have been solved for two different mammals: human and mouse. The infection of rhesus macaques (Macaca mulatta) by simian immunodeficiency virus (SIV) is the best animal model for studying HIV. In this study, the rhesus macaque CD8 (rCD8) αα homodimer was obtained and rCD8α exodomain protein crystals were successfully obtained for further structural analysis. Diffraction data were collected to a resolution of 2.4 Å. The crystal belonged to space group P212121, with unit-cell parameters a = 46.52, b = 56.28, c = 82.40 Å. These data will facilitate further studies on the structural differences between these CD8 structures and the cellular immune responses of rhesus macaque.
CD8α exodomain protein; Macaca mulatta; CD8; T-cell co-receptors
The AIDS-causing lentiviruses HIV and SIV effectively evade host immunity, and once established, infections with these viruses are only rarely controlled by immunologic mechanisms1-3. However, the initial establishment of infection in the first few days after mucosal exposure, prior to viral dissemination and massive replication, may be more vulnerable to immune control4. Here, we report that SIV vaccines that include rhesus cytomegalovirus (RhCMV) vectors5 establish indefinitely persistent, high frequency, SIV-specific effector-memory T cell (TEM) responses at potential sites of SIV replication in rhesus macaques (RM) and stringently control highly pathogenic SIVmac239 infection early after mucosal challenge. Thirteen of 24 RM receiving either RhCMV vectors alone or RhCMV vectors followed by adenovirus 5 (Ad5) vectors (vs. 0 of 9 DNA/Ad5-vaccinated RM) manifested early complete control of SIV (undetectable plasma virus), and in 12/13 of these RM, we observed long-term (≥1 year) protection characterized by: 1) occasional blips of plasma viremia that ultimately waned; 2) predominantly undetectable cell-associated viral load in blood and lymph node mononuclear cells; 3) no depletion of effector site CD4+ memory T cells; 4) no induction or boosting of SIVenv-specific antibodies (Abs); and 5) induction and then loss of T cell responses to an SIV protein (vif) not included in the RhCMV vectors. Protection correlated with the magnitude of the peak SIV-specific CD8+ T cell responses in the vaccine phase, and occurred without anamnestic T cell responses. Remarkably, long-term RhCMV vector-associated SIV control was insensitive to either CD8+ or CD4+ lymphocyte depletion, and at necropsy, cell-associated SIV was only occasionally measurable at the limit of detection with ultrasensitive assays, observations suggesting the possibility of eventual viral clearance. Thus, persistent vectors such as CMV and their associated TEM responses might significantly contribute to an efficacious HIV/AIDS vaccine.