New Drug Formulary Management and Utilization: Evidence in Sex, Race, and Ethnicity: 2019-2020.

BACKGROUND: It is well established that females and persons of racial and ethnic minorities are frequently underrepresented in clinical trials. These disparities are potentially important aspects of evidence-based formulary management and drug utilization review (DUR) processes. OBJECTIVE: The purpose of this study was to review the demographic composition of pivotal trials and post-approval study requirements for recent FDA-approved drugs, analyzing the representation of minority groups and its generalizability to the US population or corresponding disease state. METHODS: Drugs approved between July 2019 and June 2020 were identified and demographic data including race, ethnicity, and sex was extracted from their pivotal trials. Demographic data was compared to US demographics and/or the disease state demographics for the respective approved drug. RESULTS: There were a total of 85 drugs and 142 pivotal trials included in the study. Compared to the estimated US population, the minority groups with a statistically significant underrepresentation across all pivotal trials included Black or African Americans and American Indian or Alaska Natives. The Hispanic/Latinx population had a statistically significant underrepresentation in 55.4% of trials. Females had a statistically significant underrepresentation in 21.2% of trials when compared to the disease state demographics of the respective approved drug. CONCLUSION AND RELEVANCE: Persons of minorities are underrepresented in the generation of evidence of safety and efficacy for many new drugs. Formulary management and DUR offer an integrated strategic opportunity for the clinical community to formally and carefully consider the data on sex, race, and ethnicity to address disparities in health care.

Electron Acceptors Based on Cyclopentannulated Anthanthrenes.

We demonstrate the preparation of anthanthrene derivatives with fused five-membered rings. These new derivatives are prepared by a palladium catalyzed cyclopentannulation reaction from precursors derived from the low-cost dye Vat Orange 3 (4,10-dibromoanthanthrone). The new cyclopentaanthanthrene compounds possess reduced optical and electrochemical gaps (∼0.9 eV) compared to anthanthrene derivatives and are electron acceptors with lowest unoccupied molecular orbital energies from ∼ -3.4 to -3.9 eV.

Hodgkin disease relapse discovered at the time of liver transplant for acute liver failure.

Lymphoma is a recognized cause of liver damage and in rare instances presents as ALF. In such cases, the underlying malignancy is often difficult to detect. Historically, the prognosis has been poor. Cure has occasionally been achieved with chemotherapy alone. LT in this setting is controversial, but has contributed to successful outcomes, as in the case of the five-yr-old girl reported here.

Polyamine import and accumulation causes immunomodulation in macrophages engulfing apoptotic cells.

Phagocytosis of apoptotic cells, termed efferocytosis, is critical for tissue homeostasis and drives anti-inflammatory programming in engulfing macrophages. Here, we assess metabolites in naive and inflammatory macrophages following engulfment of multiple cellular and non-cellular targets. Efferocytosis leads to increases in the arginine-derived polyamines, spermidine and spermine, in vitro and in vivo. Surprisingly, polyamine accumulation after efferocytosis does not arise from retention of apoptotic cell metabolites or de novo synthesis but from enhanced polyamine import that is dependent on Rac1, actin, and PI3 kinase. Blocking polyamine import prevents efferocytosis from suppressing macrophage interleukin (IL)-1ß or IL-6. This identifies efferocytosis as a trigger for polyamine import and accumulation, and imported polyamines as mediators of efferocytosis-induced immune reprogramming.

YAP and TAZ Promote Periosteal Osteoblast Precursor Expansion and Differentiation for Fracture Repair.

In response to bone fracture, periosteal progenitor cells proliferate, expand, and differentiate to form cartilage and bone in the fracture callus. These cellular functions require the coordinated activation of multiple transcriptional programs, and the transcriptional regulators Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ) regulate osteochondroprogenitor activation during endochondral bone development. However, recent observations raise important distinctions between the signaling mechanisms used to control bone morphogenesis and repair. Here, we tested the hypothesis that YAP and TAZ regulate osteochondroprogenitor activation during endochondral bone fracture healing in mice. Constitutive YAP and/or TAZ deletion from Osterix-expressing cells impaired both cartilage callus formation and subsequent mineralization. However, this could be explained either by direct defects in osteochondroprogenitor differentiation after fracture or by developmental deficiencies in the progenitor cell pool before fracture. Consistent with the second possibility, we found that developmental YAP/TAZ deletion produced long bones with impaired periosteal thickness and cellularity. Therefore, to remove the contributions of developmental history, we next generated adult onset-inducible knockout mice (using Osx-CretetOff ) in which YAP and TAZ were deleted before fracture but after normal development. Adult onset-induced YAP/TAZ deletion had no effect on cartilaginous callus formation but impaired bone formation at 14 days post-fracture (dpf). Earlier, at 4 dpf, adult onset-induced YAP/TAZ deletion impaired the proliferation and expansion of osteoblast precursor cells located in the shoulder of the callus. Further, activated periosteal cells isolated from this region at 4 dpf exhibited impaired osteogenic differentiation in vitro upon YAP/TAZ deletion. Finally, confirming the effects on osteoblast function in vivo, adult onset-induced YAP/TAZ deletion impaired bone formation in the callus shoulder at 7 dpf before the initiation of endochondral ossification. Together, these data show that YAP and TAZ promote the expansion and differentiation of periosteal osteoblast precursors to accelerate bone fracture healing. © 2020 American Society for Bone and Mineral Research (ASBMR).

Effects of Bone Morphogenetic Protein-2 on Neovascularization During Large Bone Defect Regeneration.

Insufficient blood vessel supply is a primary limiting factor for regenerative approaches to large bone defect repair. Recombinant bone morphogenetic protein-2 (BMP-2) delivery induces robust bone formation and has been observed to enhance neovascularization, but whether the angiogenic effects of BMP-2 are due to direct endothelial cell stimulation or due to indirect paracrine signaling remain unclear. In this study, we evaluated the effects of BMP-2 delivery on vascularized bone regeneration and tested whether BMP-2 induces neovascularization directly or indirectly. We found that delivery of BMP-2 (5 µg) enhanced both bone formation and neovascularization in critically sized (8 mm) rat femoral bone defects; however, BMP-2 did not directly stimulate angiogenesis in vitro. In contrast, conditioned medium from both mesenchymal progenitor cells and osteoblasts induced endothelial cell migration in vitro, suggesting a paracrine mechanism of BMP-2 action. Consistent with this inference, codelivery of BMP-2 with endothelial colony forming cells to a heterotopic site, distant from the skeletal stem cell-rich bone marrow niche, induced ossification but had no effect on neovascularization. Taken together, these data suggest that paracrine activation of osteoprogenitor cells is an important contributor to neovascularization during BMP-2-mediated bone regeneration. Impact Statement In this study, we show that bone morphogenetic protein-2 (BMP-2) robustly induces neovascularization during tissue-engineered large bone defect regeneration, and we found that BMP-2 induced angiogenesis, in part, through paracrine signaling from osteoprogenitor cells.

Recapitulating bone development through engineered mesenchymal condensations and mechanical cues for tissue regeneration.

Large bone defects cannot form a callus and exhibit high complication rates even with the best treatment strategies available. Tissue engineering approaches often use scaffolds designed to match the properties of mature bone. However, natural fracture healing is most efficient when it recapitulates development, forming bone via a cartilage intermediate (endochondral ossification). Because mechanical forces are critical for proper endochondral bone development and fracture repair, we hypothesized that recapitulating developmental mechanical forces would be essential for large bone defect regeneration in rats. Here, we engineered mesenchymal condensations that mimic the cellular organization and lineage progression of the early limb bud in response to local transforming growth factor-ß1 presentation from incorporated gelatin microspheres. We then controlled mechanical loading in vivo by dynamically tuning fixator compliance. Mechanical loading enhanced mesenchymal condensation-induced endochondral bone formation in vivo, restoring functional bone properties when load initiation was delayed to week 4 after defect formation. Live cell transplantation produced zonal human cartilage and primary spongiosa mimetic of the native growth plate, whereas condensation devitalization before transplantation abrogated bone formation. Mechanical loading induced regeneration comparable to high-dose bone morphogenetic protein-2 delivery, but without heterotopic bone formation and with order-of-magnitude greater mechanosensitivity. In vitro, mechanical loading promoted chondrogenesis and up-regulated pericellular matrix deposition and angiogenic gene expression. In vivo, mechanical loading regulated cartilage formation and neovascular invasion, dependent on load timing. This study establishes mechanical cues as key regulators of endochondral bone defect regeneration and provides a paradigm for recapitulating developmental programs for tissue engineering.

Induction of a long-lasting AP-1 complex composed of altered Fos-like proteins in brain by chronic cocaine and other chronic treatments.

Following chronic cocaine treatment, we have found a long-lasting increase in AP-1 binding in the rat nucleus accumbens and striatum, two important targets of the behavioral effects of cocaine. This increase develops gradually over several days and remains at 50% of maximal levels 7 days after the last cocaine exposure. Supershift experiments, along with one- and two-dimensional Western blots, indicate that this chronic AP-1 complex contains at least four Fos-related antigens (FRAs), some of which display delta FosB-like immunoreactivity, that are induced selectively by chronic, but not acute, cocaine treatment. The same chronic FRAs were also induced by several different types of chronic treatments in a region-specific manner in the brain. Thus, the chronic FRAs and associated chronic AP-1 complex could mediate some of the long-term changes in gene expression unique to the chronic-treated state as opposed to the acute-treated and normal states.

Intraoperative biomechanics of lumbar pedicle screw loosening following successful arthrodesis.

Pedicle screw loosening has been implicated in recurrent back pain after lumbar spinal fusion, but the degree of loosening has not been systematically quantified in patients. Instrumentation removal is an option for patients with successful arthrodesis, but remains controversial. Here, we quantified pedicle screw loosening by measuring screw insertion and/or removal torque at high statistical power (beta = 0.02) in N = 108 patients who experienced pain recurrence despite successful fusion after posterior instrumented lumbar fusion with anterior lumbar interbody fusion (L2-S1). Between implantation and removal, pedicle screw torque was reduced by 58%, indicating significant loosening over time. Loosening was greater in screws with evoked EMG threshold under 11 mA, indicative of screw misplacement. A theoretical stress analysis revealed increased local stresses at the screw interface in pedicles with decreased difference in pedicle thickness and screw diameter. Loosening was greatest in vertebrae at the extremities of the fused segments, but was significantly lower in segments with one level of fusion than in those with two or more. CLINICAL SIGNIFICANCE: These data indicate that pedicle screws can loosen significantly in patients with recurrent back pain and warrant further research into methods to reduce the incidence of screw loosening and to understand the risks and potential benefits of instrumentation removal. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2673-2681, 2017.

Neurons that say NO.

Thirty years ago, Thomas and Pearse discovered what they termed 'solitary active cells'--neurons containing an unusually high nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-diaphorase) activity that could be detected histochemically. Although these neurons were considered as something special, an appropriate mechanism to account for their outstanding metabolism was not provided until the recent identification of neuronal NADPH-diaphorase as nitric oxide synthase. This simple histochemical method now allows the precise anatomical localization of the neurons generating the exotic messenger molecule nitric oxide. This article reviews the functional implications that arise from our new knowledge of the anatomy of the nitric oxide signal transduction pathway in the nervous system. The widespread distribution of this system indicates that for those interested in cellular communication nitric oxide is a gas to study.

Tyrosine hydroxylase containing neurons lacking aromatic amino acid decarboxylase in the hamster brain.

We have recently described populations of tyrosine hydroxylase-immunoreactive neurons in the hamster brain in regions not known to contain catecholamine cell bodies. In the present study, the nature of the tyrosine hydroxylase immunoreactivity in the hamster brain was determined. In addition, these tyrosine hydroxylase-immunoreactive cell groups were examined for their ability to express aromatic amino acid decarboxylase. Immunohistochemistry with two different antibodies to tyrosine hydroxylase identified immunoreactive cell bodies in regions known to contain catecholamine neurons, including the substantia nigra and locus ceruleus. In addition, tyrosine hydroxylase-immunoreactive neurons were observed in other regions, including the basal forebrain, inferior colliculus, lateral parabrachial nucleus, and dorsal motor nucleus of the vagus. Western blotting indicated that hamster brain contained only one immunoreactive molecule, very similar in size to rat tyrosine hydroxylase. Thus it is likely that the immunohistochemical studies stained authentic hamster tyrosine hydroxylase. Indeed, in situ hybridization studies using a synthetic oligonucleotide probe against tyrosine hydroxylase mRNA resulted in specific and heavy labelling of these novel tyrosine hydroxylase-immunoreactive neurons. When adjacent sections were stained with antibodies to aromatic amino acid decarboxylase, known catecholamine cell groups were stained. However, the novel tyrosine hydroxylase cell groups did not display any aromatic amino acid decarboxylase immunoreactivity. These results suggest that neurons are present in the hamster brain that are able to hydroxylate tyrosine to L-DOPA, but that lack the ability to decarboxylate aromatic amino acids to produce dopamine or other catecholamines.

Cocaine-induced Fos expression in rat striatum is blocked by chloral hydrate or urethane.

Anesthetics used in electrophysiological studies alter the effects of cocaine and amphetamine on neural activity in the striatum. However, the mechanism underlying this alteration has not been established. In the present study, we examined the effects of anesthetics on cocaine-induced neural activity in the striatum. We first assayed the ability of 20 mg/kg cocaine to induce Fos expression in the striatum following pretreatment with 400 mg/kg chloral hydrate or 1.3 g/kg urethane, two of the most commonly used anesthetics for in vivo electrophysiology. Chloral hydrate blocked, while urethane strongly attenuated cocaine-induced Fos expression without affecting basal levels of expression. We then examined dopaminergic and glutamatergic mechanisms for anesthetic effects on cocaine-induced Fos expression. Chloral hydrate and urethane did not attenuate basal or cocaine-induced increases of dopamine levels as assessed by microdialysis in dorsal striatum. In contrast, chloral hydrate attenuated glutamatergic neurotransmission as assessed by microdialysis in the presence of the glutamate transport blocker L-trans-pyrrolidone-2,4-dicarboxylic acid. Chloral hydrate attenuated basal levels of glutamate by 70%, while cocaine had no effect on glutamate levels. Since glutamate levels were tetrodotoxin-sensitive, the majority of glutamate measured in our assay was by synaptic release. To assess a causal role for a reduction of glutamatergic neurotransmission in anesthetic effects on cocaine-induced Fos expression, we injected the glutamate receptor agonists AMPA and NMDA into the dorsal striatum of chloral hydrate-anesthetized rats. The glutamate receptor agonists partially reinstated cocaine-induced Fos expression in anesthetized rats. We conclude anesthetics attenuate cocaine-induced neuronal activity by reducing glutamatergic neurotransmission.

Kappa-opioid receptor activation prevents alterations in mesocortical dopamine neurotransmission that occur during abstinence from cocaine.

In vivo microdialysis was used to characterize basal dopamine dynamics and cocaine-evoked dopamine levels in the medial prefrontal cortex of male Sprague-Dawley rats that had previously received once daily injections of cocaine (days 1-5; 20mg/kg, i.p.) in combination with the selective kappa-opioid receptor agonist U-69593 (days 3-5; 0.32mg/kg, s.c.) or its vehicle. The influence of these treatments on [3H]dopamine uptake in medial prefrontal cortex synaptosomes was also determined. Three days following the cessation of drug treatment, animals with prior history of cocaine administration exhibited enhanced psychomotor stimulation in response to a subsequent cocaine challenge. This effect was not apparent in animals that had previously received the cocaine treatment regimen in combination with the kappa-opioid receptor agonist U-69593. Cocaine challenge increased prefrontal dopamine levels in all pretreatment groups, but cocaine-pre-exposed animals had lower cocaine-evoked dopamine levels and higher basal in vivo extraction fraction, indicative of an increase in basal dopamine uptake relative to controls. Pretreatment with U-69593 prevented these effects of cocaine. Measurement of [3H]dopamine uptake in synaptosomes revealed a significant increase in uptake three days after the cessation of cocaine treatment. No increase in uptake was observed in animals that had received the cocaine treatment regimen in combination with U-69593. These results demonstrate that the early phase of abstinence from cocaine is associated with marked alterations in medial prefrontal cortex dopamine neurotransmission and that these neuroadaptations are prevented by the activation of kappa-opioid receptors. Furthermore, they raise the possibility that mesocortical dopamine neurons may be an important neural substrate upon which kappa-opioid agonists act to prevent the development of cocaine-induced behavioral sensitization.

Histochemical characterization of neuronal NADPH-diaphorase.

We examined the properties of neuronal NADPH-diaphorase in sections of rat striatum, using histochemical procedures. NADPH-diaphorase histochemistry stained discrete populations of central neurons and provided a Golgi-like image of the neurons exhibiting this activity. The NADPH-diaphorase reaction appeared to be enzyme catalyzed, since it was abolished by pre-treatment with proteases, heat, and acid or alkaline denaturation. Under anaerobic conditions, any tetrazolium salt with a redox potential more positive than NADPH could be reduced by the enzyme. NADPH-diaphorase activity was sensitive to inhibition by sulfhydryl reagents but was unaffected by metal chelators, superoxide dismutase, and catalase. Therefore, the enzyme is unlikely to be a metalloenzyme or to reduce tetrazoliums by producing superoxide anions or hydrogen peroxide. Various analogues of beta-NADPH could be used by the enzyme; however, beta-NADH, which can be used by DT-diaphorase, was ineffective. The enzyme was also resistant to dicumarol, an inhibitor of DT-diaphorase activity. Electron microscopy indicated that the NADPH-diaphorase reaction resulted in staining of various membranous organelles. We conclude that neuronal NADPH-diaphorase is a membrane-bound enzyme distinct from DT-diaphorase and other known enzymes with diaphorase activity. The histochemical characteristics presented here should now enable meaningful biochemical studies of neuronal NADPH-diaphorase to be undertaken.

Time-dependent changes in extinction behavior and stress-induced reinstatement of drug seeking following withdrawal from heroin in rats.

RATIONALE AND OBJECTIVES: Footshock stress reliably reinstates heroin seeking in rats, but the time course of the development of this effect following drug withdrawal is not known. Here we studied the effect of intermittent footshock stress on reinstatement of heroin seeking following different withdrawal periods (1-66 days). We also studied whether changes in corticotropin-releasing factor (CRF) mRNA in the central nucleus of the amygdala (CeA) and the bed nucleus of the stria terminalis (BNST) are correlated with this reinstatement after 1 day and 6 days of heroin withdrawal. METHODS: Rats were trained to self-administer heroin (9 h/day; 0.1 mg/kg per infusion) for 10 days. Tests for extinction behavior and footshock-induced reinstatement of heroin seeking were then conducted after 1, 6, 12, 25, or 66 days of heroin withdrawal. On the test day, rats were given five to ten 60-min extinction sessions until they reached the extinction criterion of less than 15 responses per 60 min on the lever previously associated with heroin. Rats were then exposed to intermittent foot-shock (0.8 mA; 10 min), and lever-pressing behavior was recorded for 120 min. RESULTS: Reinstatement of lever-pressing behavior by footshock followed an inverted U-shaped curve with maximal responding after 6 days and 12 days of heroin withdrawal. Surprisingly, foot-shock did not reinstate lever-pressing behavior on day 1 of withdrawal. Lever pressing during extinction, prior to exposure to footshock, also followed an inverted U-shaped curve, with higher responding after 6, 12, and 25 days of heroin withdrawal. Finally, compared with control groups not exposed to shock, CRF mRNA levels in response to footshock were increased in the CeA (day 1 of withdrawal) and the dorsal BNST (day 1 and day 6), but not in the ventral BNST. CONCLUSIONS: The duration of the heroin withdrawal period is an important factor in the manifestation of (1) footshock stress-induced reinstatement of heroin seeking and (2) extinction of the heroin-reinforced behavior. Finally, the time-dependent changes in footshock stress-induced reinstatement following withdrawal from heroin were not correlated with alterations in CRF mRNA in the CeA and BNST.

Novel transcription factors are induced by chronic cocaine treatment.

There are many other transcription factors that are regulated by cocaine, and there are several nontranscriptional mechanisms of regulating protein levels as well. However, the induction of the chronic AP-1 complex and the chronic Fras provides a mechanism capable of underlying long-lasting alterations in gene expression following chronic cocaine treatment. We hypothesize that while the well-known acute effects of cocaine are occurring, the acutely induced cAMP pathway, possibly in conjunction with other second messenger pathways, is also inducing two general types of alterations in gene expression. 1. Acutely induced significant alterations in gene expression are rapidly induced and relatively short-lived. These alterations in gene expression would allow the cell to rapidly adapt to cocaine in its environment without committing to long-term changes. 2. Additional alterations in gene expression are induced by each acute administration during chronic cocaine exposure. These latter alterations are gradually induced and long-lasting. With each acute activation, the level of induction of these latter longer-lasting alterations in gene expression would be relatively small and insignificant compared to the rapidly induced acute alterations in gene expression. Therefore, with each acute administration, the cell does not commit itself to long-term alterations. However, these small but longer lasting changes in gene expression would accumulate with each acute administration during chronic cocaine treatment, similar to the accumulation of the chronic AP-I complex (FIG. 4). The cumulative alterations which occur during repeated administrations eventually reach a level where they produce significant and long-lasting alterations in gene expression different from those induced acutely. This incremental induction of long-lasting alterations in gene expression would allow the cell to gradually commit to long-term alterations for the purpose of adapting to repeated long-term exposure to cocaine in its environment. These long-lasting alterations in gene expression may underlie the gradual induction of persistent changes in protein levels, neuronal signaling, and related behaviors. This hypothesis allows for the different adaptations observed following acute versus chronic cocaine administration at the biochemical, physiological, and behavioral levels. We hope these investigations provide ideas for specific pharmacological blockade or reversal of the long-term, versus short-term, alterations in gene expression that lead to addiction, without interfering with the animal's or human's ability to experience reward. This would be important for ensuring both efficacy and compliance.

Cocaine and the AP-1 transcription factor complex.

Cocaine addition in humans develops gradually with repeated administrations and persists long after cocaine has cleared the body. The mechanisms underlying this persistent form of neuroplasticity are not understood and can involve both structural and biochemical mechanisms. The long time course for cocaine addiction in humans and for development of cocaine self-administration in animal models suggest the involvement of alterations in gene expression leading to altered signaling in the brain. In the striatum (Str) and nucleus accumbens (NAc) of rats. Pretreatment with repeated cocaine administrations downregulates the induction of various immediate early genes (IEGs) by a subsequent acute challenge with cocaine. Some of these downregulated IEGs encode Fos-related components of the activator protein-1 (AP-1) complex, which is likely re regulate a number of genes important for neuronal function. Interestingly, repeated cocaine administration induces novel delta FosB-related proteins (called chronic Fos-related antigens (Fras)) in the NAc and Str that replace the downregulated isoforms of Fos. Unlike the acutely induced, short-lasting isoforms of Fos and FosB, the chronic Fras persist long after the last cocaine administration. The known form of delta FosB per se lacks the domain required to activate transcription. If the chronic Fras are similar in structure to delta FosB, then the induction of chronic Fras likely leads to a blockade of AP-1-dependent transcription resulting in altered gene expression. We presently purifying the chronic Fras to obtain amino acid sequence in order to directly examine our hypothesis about the effects of repeated cocaine administration on AP-1 dependent transcription and gene expression in the brain

Induction of the c-fos proto-oncogene in rat amygdala during unconditioned and conditioned fear.

Induction of the nuclear proto-oncogene c-fos in rat amygdala was investigated 30-40 min following the presentation of mild footshocks (unconditioned fear) or of contextual cues associated with similar footshocks 24 h earlier (conditioned fear). Initially, it was found that handling rats for the first time elevated c-fos mRNA levels, but this response could be blocked completely by repeated handling. Unconditioned and conditioned fear both elevated amygdala c-fos mRNA dramatically above control levels.

Citrulline in the rat brain: immunohistochemistry and coexistence with NADPH-diaphorase.

The presence in the brain of the urea cycle intermediate citrulline in the absence of a complete urea cycle has never been adequately explained. In an attempt to clarify this problem, we developed antibodies to citrulline and determined the distribution of citrulline-immunoreactivity in fixed sections of rat brain using immunoperoxidase and indirect immunofluorescence techniques. Citrulline-positive neurons were found to have a restricted distribution within the brain. A few cells were present in the cortex and corpus callosum. A large population of strongly stained cells was diffusely scattered throughout the striatum, nucleus accumbens and olfactory tubercle. Less strongly stained cells were detected in the supraoptic and paraventricular nuclei of the hypothalamus, the dorsal raphe, and the laterodorsal and pedunculopontine tegmental nuclei of the pons. The citrulline-immunoreactive cells were similar to those previously shown to contain NADPH-diaphorase activity, and double staining experiments indicated that citrulline-immunoreactivity was present in a subpopulation of NADPH-diaphorase-positive neurons. We have recently identified NADPH-diaphorase as a nitric oxide synthase. Thus the presence of citrulline in these cells suggests that it is formed within the brain as a coproduct during nitric oxide formation from arginine.

Impact of combination therapies on HIV risk perceptions and sexual risk among HIV-positive and HIV-negative gay and bisexual men.

The availability of improved HIV treatments may prompt reduced concern about HIV and sexual risk. Gay and bisexual men (N = 554, 17% HIV-positive) completed measures of treatment attitudes, sexual risk, and assumptions regarding the infectiousness of sexual partners. A substantial minority reported reduced HIV concern related to treatment advances. Reduced HIV concern was an independent predictor of sexual risk, particularly among HIV-positive men. In response to hypothetical scenarios describing sex with an HIV-positive partner, participants rated the risk of unprotected sex to be lower if the partner was taking combination treatments and had an undetectable viral load, relative to scenarios with a seropositive partner not taking combination treatments. Prevention efforts must address attitudinal shifts prompted by recent treatment successes, stressing the continued importance of safer sex, and that an undetectable viral load does not eliminate infection risks.