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BACKGROUND Human immunodeficiency virus (HIV) infection and antiretroviral therapy (ART) are associated with bone loss leading to increased fracture rate among HIV-infected individuals. ART-induced bone loss is most intense within the first 48 weeks of therapy, providing a window for prophylaxis with long-acting antiresorptives. METHODS In a phase 2, double-blind, placebo-controlled trial, we randomized 63 nonosteoporotic, ART-naive adults with HIV initiating ART with atazanavir/ritonavir + tenofovir/emtricitabine to a single zoledronic acid (ZOL) infusion (5 mg) vs placebo to determine the efficacy of ZOL in mitigating ART-induced bone loss. Plasma bone turnover markers and bone mineral density (BMD) were performed at weeks 0, 12, 24, and 48 weeks. Primary outcome was change in C-terminal telopeptide of collagen at 24 weeks. Repeated-measures analyses using mixed linear models were used to estimate and compare study endpoints. RESULTS The ZOL arm had a 65% reduction in bone resorption relative to the placebo arm at 24 weeks (0.117 ng/mL vs 0.338 ng/mL; P < .001). This effect of ZOL occurred as early as 12 weeks (73% reduction; P < .001) and persisted through week 48 (57% reduction; P < .001). The ZOL arm had an 8% higher lumbar spine BMD at 12 weeks relative to the placebo arm (P = .003), and remained 11% higher at 24 and 48 weeks. Similar trends were observed in the hip and femoral neck. CONCLUSIONS A single dose of ZOL administered at ART initiation prevented ART-induced bone loss through the first 48 weeks of ART, the period when ART-induced bone loss is most pronounced. Validation of these results in larger multicenter randomized clinical trials is warranted. CLINICAL TRIALS REGISTRATION NCT01228318.

Intracellular nucleoside reverse transcriptase inhibitor (NRTI) concentrations are associated with plasma HIV-1 response. Coadministration of protease inhibitors with NRTIs can affect intra-cellular concentrations due to protease inhibitor inhibition of efflux transporters. Tenofovir-diphosphate (TFV-DP) concentrations within peripheral blood mononuclear cells were compared among individuals receiving either atazanavir or darunavir-based regimens. There was a trend towards higher TFV-DP concentrations among women and among participants receiving atazanavir. TFV-DP intracellular concentrations were positively associated with undetectable plasma HIV-1 RNA.

A growing body of evidence suggests that gonadal steroids such as estradiol (E2) alter neural responses not only in brain regions associated with reproductive behavior but also in sensory areas. Because catecholamine systems are involved in sensory processing and selective attention, and because they are sensitive to E2 in many species, they may mediate the neural effects of E2 in sensory areas. Here, we tested the effects of E2 on catecholaminergic innervation, synthesis and activity in the auditory system of white-throated sparrows, a seasonally breeding songbird in which E2 promotes selective auditory responses to song. Non-breeding females with regressed ovaries were held on a winter-like photoperiod and implanted with silastic capsules containing either no hormone or E2. In one hemisphere of the brain, we used immunohistochemistry to quantify fibers immunoreactive for tyrosine hydroxylase or dopamine beta-hydroxylase in the auditory forebrain, thalamus and midbrain. E2 treatment increased catecholaminergic innervation in the same areas of the auditory system in which E2 promotes selectivity for song. In the contralateral hemisphere we quantified dopamine, norepinephrine and their metabolites in tissue punches using HPLC. Norepinephrine increased in the auditory forebrain, but not the midbrain, after E2 treatment. We found that evidence of interhemispheric differences, both in immunoreactivity and catecholamine content that did not depend on E2 treatment. Overall, our results show that increases in plasma E2 typical of the breeding season enhanced catecholaminergic innervation and synthesis in some parts of the auditory system, raising the possibility that catecholamines play a role in E2-dependent auditory plasticity in songbirds.

Social behaviors such as courtship, parenting, and aggression depend primarily on two factors: a social signal to trigger the behavior, and a hormonal milieu that facilitates or permits it. Gonadal steroids may alter the valence or perceived context of the signal so that the same pheromone, vocalization, or visual display may elicit very different responses depending on the receiver's plasma hormone level. The neural processes underlying this phenomenon, however, are not well understood. Here, we describe how hormones modulate neural responses to social signals in female white-throated sparrows listening to recordings of male song. While manipulating levels of the ovarian steroid estradiol, we mapped and quantified sound-induced expression of the immediate early gene egr-1 in nine brain regions that constitute a social behavior network in vertebrates. In most regions of interest, hearing male song induced more expression than hearing tones or silence, and this selectivity for song was seen only in birds with estradiol levels typical of the breeding season. In females with regressed ovaries and no exogenous estradiol, neural responses were selective for song over tones only in the lateral portion of the ventromedial hypothalamus, not in the rest of the network. Because the effects of hormone treatment on neural responses are not identical in each region, the overall pattern of activation across the network changes with estradiol level and thus with season and breeding context. Our results demonstrate a possible mechanism by which gonadal steroids may alter the processing of social signals and affect social decision-making. J. Comp. Neurol. 511:173–186, 2008. © 2008 Wiley-Liss, Inc.

SUMMARY During social interactions, apteronotid electric fish modulate their electric organ discharges (EODs) to produce transient communication signals known as chirps. Chirps vary widely across species and sex in both number and structure. In Apteronotus leptorhynchus, males chirp far more than females and their chirps have greater frequency modulation than those of females. High-frequency chirps are produced by males most often in response to female-like electric signals. As such, they have been hypothesized to function in courtship. The more common low-frequency chirps, produced by both males and females in response to same-sex signals, are hypothesized to function as aggressive signals. To determine whether the two chirp types in the closely related Apteronotus albifrons have similar functions, we stimulated chirping in male and female A. leptorhynchus and A. albifrons with playbacks simulating the EODs of same-sex versusopposite-sex conspecifics. As in A. leptorhynchus, male and female A. albifrons produced low-frequency chirps most often to same-sex signals. Unlike A. leptorhynchus, however, A. albifrons also produced more high-frequency chirps to same-sex stimuli than to opposite-sex stimuli. This suggests that high-frequency chirps in A. albifrons,unlike those in A. leptorhynchus, may not function as courtship signals and that the function of similar chirp types has diversified in Apteronotus. Examples such as this, in which the function of a communication signal has changed in closely related species, are rare. The electrocommunication signals of apteronotids may thus provide a remarkable opportunity to investigate the evolutionary interactions of signal structure and function.

HIV infection is associated with high rates of osteopenia and osteoporosis, but the mechanisms involved are unclear. We recently reported that bone loss in the HIV transgenic rat model was associated with upregulation of B cell expression of the key osteoclastogenic cytokine receptor-activator of NF-κB ligand (RANKL), compounded by a simultaneous decline in expression of its physiological moderator, osteoprotegerin (OPG). To clinically translate these findings we performed cross-sectional immuno-skeletal profiling of HIV-uninfected and antiretroviral therapy-naïve HIV-infected individuals. Bone resorption and osteopenia were significantly higher in HIV-infected individuals. B cell expression of RANKL was significantly increased, while B cell expression of OPG was significantly diminished, conditions favoring osteoclastic bone resorption. The B cell RANKL/OPG ratio correlated significantly with total hip and femoral neck bone mineral density (BMD), T- and/or Z-scores in HIV infected subjects, but revealed no association at the lumbar spine. B cell subset analyses revealed significant HIV-related increases in RANKL-expressing naïve, resting memory and exhausted tissue-like memory B cells. By contrast, the net B cell OPG decrease in HIV-infected individuals resulted from a significant decline in resting memory B cells, a population containing a high frequency of OPG-expressing cells, concurrent with a significant increase in exhausted tissue-like memory B cells, a population with a lower frequency of OPG-expressing cells. These data validate our pre-clinical findings of an immuno-centric mechanism for accelerated HIV-induced bone loss, aligned with B cell dysfunction., Author Summary HIV infection causes significant bone loss and skeletal deterioration, leading to fractures that are often devastating and incur significant financial burden on patients and their families. HIV-infected individuals have up to a five-fold higher risk of bone fractures, and the increasing average age of people living with HIV/AIDS has triggered fears of an impending epidemic of bone fractures in this population. Antiretroviral therapy, used to manage HIV infection, fails to prevent, but rather paradoxically accelerates skeletal decline. The underlying mechanisms of HIV-induced bone loss are poorly understood. The aim of this study was to clarify the mechanisms of bone loss in HIV-infected patients, in an effort to better understand how bone loss and fractures occur, and consequently how it can be prevented in this population. The cytokine RANKL (Receptor Activator of Nuclear Factor kappa-B Ligand) helps induce bone loss. We show that RANKL expression was increased in immune cells in HIV-infected individuals. Another cytokine, osteoprotegerin (OPG), counteracts the activity of RANKL, and therefor helps prevent bone loss. OPG expression by the same immune cells was decreased in HIV-infected individuals. We conclude that disrupted immune cell expression of RANKL and OPG in HIV-infected patients contributes to bone loss.

Catecholaminergic (CA) neurons innervate sensory areas and affect the processing of sensory signals. For example, in birds, CA fibers innervate the auditory pathway at each level, including the midbrain, thalamus, and forebrain. We have shown previously that in female European starlings, CA activity in the auditory forebrain can be enhanced by exposure to attractive male song for one week. It is not known, however, whether hearing song can initiate that activity more rapidly. Here, we exposed estrogen-primed, female white-throated sparrows to conspecific male song and looked for evidence of rapid synthesis of catecholamines in auditory areas. In one hemisphere of the brain, we used immunohistochemistry to detect the phosphorylation of tyrosine hydroxylase (TH), a rate-limiting enzyme in the CA synthetic pathway. We found that immunoreactivity for TH phosphorylated at serine 40 increased dramatically in the auditory forebrain, but not the auditory thalamus and midbrain, after 15 min of song exposure. In the other hemisphere, we used high pressure liquid chromatography to measure catecholamines and their metabolites. We found that two dopamine metabolites, dihydroxyphenylacetic acid and homovanillic acid, increased in the auditory forebrain but not the auditory midbrain after 30 min of exposure to conspecific song. Our results are consistent with the hypothesis that exposure to a behaviorally relevant auditory stimulus rapidly induces CA activity, which may play a role in auditory responses.

A nucleoside reverse transcriptase inhibitor (NRTI) backbone is a recommended component of standard highly active antiretroviral therapy (sHAART). However, long-term NRTI exposure can be limited by toxicities. NRTI class-sparing alternatives are warranted in select patient populations. This is a 48-week single-center, open-label pilot study in which 60 HIV-infected adults with plasma HIV-1 RNA (50 copies/ml) on sHAART were randomized (2:1) to lopinavir/ritonavir (LPV/r) 400/100 mg BID+raltegravir (RAL) 400 mg BID switch (LPV-r/RAL arm) or to continue on sHAART. The primary endpoint was the proportion of subjects with HIV-RNA50 copies/ml at week 48. Secondary efficacy and immunologic and safety endpoints were evaluated. Demographics and baseline lipid profile were similar across arms. Mean entry CD4 T cell count was 493 cells/mm(3). At week 48, 92% [95% confidence interval (CI): 83-100%] of the LPV-r/RAL arm and 88% (95% CI: 75-100%) of the sHAART arm had HIV-RNA50 copies/ml (p=0.70). Lipid profile (mean ± SEM, mg/dl, LPV-r/RAL vs. sHAART) at week 24 was total-cholesterol 194 ± 5 vs. 176 ± 9 (p=0.07), triglycerides 234 ± 30 vs. 133 ± 27 (p=0.003), and LDL-cholesterol 121 ± 6 vs. 110 ± 8 (p=0.27). There were no serious adverse events (AEs) in either arm. Regimen change occurred in three LPV-r/RAL subjects (n=1, due to LPV-r/RAL-related AEs) vs. 0 in sHAART. There were no differences between arms in bone mineral density, total body fat composition, creatinine clearance, or CD4 T cell counts at week 48. In virologically suppressed patients on HAART, switching therapy to the NRTI-sparing LPV-r/RAL combination produced similar sustained virologic suppression and immunologic profile as sHAART. AEs were comparable between arms, but the LPV-r/RAL arm experienced higher triglyceridemia.

Because no organism lives in an unchanging environment, sensory processes must remain plastic so that in any context, they emphasize the most relevant signals. As the behavioral relevance of sociosexual signals changes along with reproductive state, the perception of those signals is altered by reproductive hormones such as estradiol (E2). We showed previously that in white-throated sparrows, immediate early gene responses in the auditory pathway of females are selective for conspecific male song only when plasma E2 is elevated to breeding-typical levels. In this study, we looked for evidence that E2-dependent modulation of auditory responses is mediated by serotonergic systems. In female nonbreeding white-throated sparrows treated with E2, the density of fibers immunoreactive for serotonin transporter innervating the auditory midbrain and rostral auditory forebrain increased compared with controls. E2 treatment also increased the concentration of the serotonin metabolite 5-HIAA in the caudomedial mesopallium of the auditory forebrain. In a second experiment, females exposed to 30 min of conspecific male song had higher levels of 5-HIAA in the caudomedial nidopallium of the auditory forebrain than birds not exposed to song. Overall, we show that in this seasonal breeder, (a) serotonergic fibers innervate auditory areas; (b) the density of those fibers is higher in females with breeding-typical levels of E2 than in nonbreeding, untreated females; and (c) serotonin is released in the auditory forebrain within minutes in response to conspecific vocalizations. Our results are consistent with the hypothesis that E2 acts via serotonin systems to alter auditory processing.

A growing body of evidence suggests that gonadal steroids such as estradiol (E2) alter neural responses not only in brain regions associated with reproductive behavior but also in sensory areas. Because catecholamine systems are involved in sensory processing and selective attention, and because they are sensitive to E2 in many species, they may mediate the neural effects of E2 in sensory areas. Here, we tested the effects of E2 on catecholaminergic innervation, synthesis and activity in the auditory system of white-throated sparrows, a seasonally breeding songbird in which E2 promotes selective auditory responses to song. Non-breeding females with regressed ovaries were held on a winter-like photoperiod and implanted with silastic capsules containing either no hormone or E2. In one hemisphere of the brain, we used immunohistochemistry to quantify fibers immunoreactive for tyrosine hydroxylase or dopamine beta-hydroxylase in the auditory forebrain, thalamus and midbrain. E2 treatment increased catecholaminergic innervation in the same areas of the auditory system in which E2 promotes selectivity for song. In the contralateral hemisphere we quantified dopamine, norepinephrine and their metabolites in tissue punches using HPLC. Norepinephrine increased in the auditory forebrain, but not the midbrain, after E2 treatment. We found that evidence of interhemispheric differences, both in immunoreactivity and catecholamine content that did not depend on E2 treatment. Overall, our results show that increases in plasma E2 typical of the breeding season enhanced catecholaminergic innervation and synthesis in some parts of the auditory system, raising the possibility that catecholamines play a role in E2-dependent auditory plasticity in songbirds.

Sex steroids facilitate dramatic changes in behavioral responses to sociosexual signals and are increasingly implicated in the sensory processing of those signals. Our previous work demonstrated that in female white-throated sparrows, which are seasonal breeders, genomic responses in the auditory forebrain are selective for conspecific song over frequency-matched tones only when plasma estradiol (E2) reaches breeding levels. Here, we sought to map this E2-dependent selectivity in the best-studied area of the auditory forebrain, the caudomedial nidopallium (NCM). Nonbreeding females with low endogenous levels of E2 were treated with E2 or a placebo and exposed to conspecific song, tones, or no sound playback. Immunoreactive protein product of the immediate early gene zenk (egr-1) was then quantified within seven distinct subregions, or domains, of NCM. We report three main findings: (1) regardless of hormone treatment, the zenk response is significantly higher in dorsal than in ventral NCM, and higher in medial than in lateral NCM; (2) E2-dependent selectivity of the response is limited to the rostral and medial domains of NCM; in the more caudal domains, song induces more zenk expression than tones regardless of hormone treatment; (3) even when no sound stimuli were presented, E2 treatment significantly increased zenk expression in the rostral, but not the caudal, domains of NCM. Together, the latter two findings suggest that E2-dependent plasticity in NCM is concentrated in rostral NCM, which is hodologically and neurochemically distinct from caudal NCM. Activity in rostral NCM may therefore be seasonally regulated in this species. © 2009 Wiley Periodicals, Inc. Develop Neurobiol 2010

White-throated sparrows (Zonotrichia albicollis) exhibit a behavioral polymorphism that segregates with a plumage marker. Individuals with a white stripe (WS) on the crown engage in an aggressive strategy that involves more singing, whereas individuals with a tan stripe (TS) sing less and engage in more parental care. Previous work has shown that plasma levels of gonadal steroids differ between the morphs in both sexes, suggesting a hormonal mechanism for the polymorphic behavior in this species. Here, we eliminated morph differences in plasma levels of testosterone (T) in males and estradiol (E2) in females in order to test whether morph differences in behavior would be similarly eliminated. Males and females in non-breeding condition were treated with T or E2, respectively, so that plasma levels in the treated groups were high and equal between the WS and TS morphs. We found that despite hormone treatment, WS and TS birds differed with respect to singing behavior. WS males sang more in response to song playback than did TS males, and WS females exhibited more spontaneous song than TS females. We also found that WS males gave more chip calls, which are often used in contexts of territorial aggression. Overall, these results suggest that WS birds engage in more territorial vocalization, particularly song, than do TS birds, even when T or E2 levels are experimentally equalized. This behavioral difference may therefore be driven by other factors, such as steroid metabolism, receptor expression or function, or steroid-independent neurotransmitter systems.