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.

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.

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).

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.

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.

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.

Rapid rise in incidence of Irish paediatric inflammatory bowel disease.

AIMS: To describe the change in incidence of paediatric inflammatory bowel disease (IBD) observed at the National Centre for Paediatric Gastroenterology, Hepatology and Nutrition, and to determine whether the presenting disease phenotype and disease outcomes have changed during the past decade. METHODS: The annual incidence of IBD in Irish children aged <16 years was calculated for the years 2000-2010. Two subsets of patients, group A (diagnosed between 1 January 2000 and 31 December 2001), and group B (diagnosed between 1 January and 31 December 2008) were phenotyped according to the Paris Classification. Phenotype at diagnosis and 2-year follow-up were then compared. RESULTS: 406 new cases of IBD were identified. The incidence was 2.5/100 000/year in 2001, 7.3 in 2008 and 5.6 in 2010, representing a significant increase in the number of new cases of Crohn's disease (CD) and ulcerative colitis (UC). There were 238 cases of CD; 129 of UC; and 39 of IBD unclassified. Comparing groups A and B, no differences were found in disease location at diagnosis or, for CD, in its behaviour. CONCLUSIONS: There has been a substantial and sustained increase in the incidence of childhood UC and CD in Ireland over a relatively short period of time. However, disease phenotype at diagnosis has not changed. At 2 years follow-up, CD appears to progress less frequently than in some neighbouring countries. These variations remain unexplained. Prospective longitudinal studies will help to elucidate further the epidemiology of childhood IBD.

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.

A health risk assessment for fluoride in Central Europe.

Like many elements, fluorine (which generally occurs in nature as fluoride) is beneficial to human health in trace amounts, but can be toxic in excess. The links between low intakes of fluoride and dental protection are well known; however, fluoride is a powerful calcium-seeking element and can interfere with the calcified structure of bones and teeth in the human body at higher concentrations causing dental or skeletal fluorosis. One of the main exposure routes is via drinking water and the World Health Organisation currently sets water quality guidelines for the element. In Central Europe, groundwater resources that exceed the guideline value of 1.5 mg l-1 are widespread and effects on health of high fluoride in water have been reported. The aim of the current project was to develop a geographic information system (GIS) to aid the identification of areas where high-fluoride waters and fluorosis may be a problem; hence, where water treatment technologies should be targeted. The development of the GIS was based upon the collation and digitisation of existing information relevant to fluoride risk in Ukraine, Moldova, Hungary and Slovakia assembled for the first time in a readily accessible form. In addition, geochemistry and health studies to examine in more detail the relationships between high-fluoride drinking waters and health effects in the population were carried out in Moldova and Ukraine demonstrating dental fluorosis prevalence rates of 60-90% in adolescents consuming water containing 2-7 mg l-1 fluoride.

Orthopaedic manifestations of the hip in haemophilia--impaction bone grafting used in revision total hip arthroplasty in a haemophilic patient.

The condition of haemophilia presents many challenges to the orthopaedic surgeon. Two such challenges include the haemophilic pseudotumour, a rare complication occurring in 1-2% of haemophiliacs, and the phenomenon of aseptic loosening of arthroplasty implants. This report presents a case involving the separate management of a pseudotumour and revision hip arthroplasty in the same hip using impaction bone grafting.

Mercury levels and relationships in water, sediment, and fish tissue in the Willamette Basin, Oregon.

In Oregon's Willamette River Basin (the Basin), health advisories currently limit consumption of fish that have accumulated methylmercury (MeHg) to levels posing a significant human health risk. These advisories created the requirement for a mercury total maximum daily load for the Basin, which required a greater understanding of the behavior, distribution, and levels of mercury and MeHg in the Basin. In 2002, the Oregon Department of Environmental Quality initiated a study to measure (using ultraclean techniques) mercury and MeHg levels in water, sediment, and fish samples collected throughout the Basin. Results from the Middle Fork (nominal background) suggested that naturally occurring surface-water concentrations of mercury and MeHg would on an annual average basis be expected in the range of 0.5 to 1.0 and 0.04 to 0.06 ng L(-1), respectively. Concentrations in the Coast Fork (Cottage Grove), which were markedly higher, are likely the result of historical mining discharges. The possibility exists that wetlands alone could contribute the dissolved MeHg levels (approximately 0.04 ng L(-1)) observed in the Main Stem. Mercury levels in sediment were similar, and near background, in the Main Stem, Coast Fork (Row River), and Middle Fork but significantly increased in the Coast Fork (Cottage Grove). Fish tissue mercury levels were typically highest in piscivorous and lowest in invertivorous species but highest in the Coast Fork (Cottage Grove). In the Coast Fork and Cottage Grove Reservoir, discharges from historical mercury mining activities appear to have significantly impacted water, sediment, and fish tissue levels; however these impacts do not appear to extend into the Main Stem. Basinwide mercury data are at present too spottily distributed to determine whether significant mercury point sources exist along the Main Stem.

Neuroadaptations of total levels of adenylate cyclase, protein kinase A, tyrosine hydroxylase, cdk5 and neurofilaments in the nucleus accumbens and ventral tegmental area do not correlate with expression of sensitized or tolerant locomotor responses to cocaine.

Neuroadaptations induced by high-dose cocaine treatment have been hypothesized to persist after the cessation of drug treatment and mediate the expression of sensitization and tolerance to cocaine. We looked for evidence of these neuroadaptations in rats receiving more modest behaviorally effective cocaine treatments. Rats were exposed to either a sensitizing regimen of seven once-daily injections of 15 mg/kg cocaine or a tolerance-producing regimen involving a continuous infusion of the same daily dose. We assessed enzyme activity levels of protein kinase A and adenylate cyclase, and protein levels of tyrosine hydroxylase, cdk5 and neurofilaments in the nucleus accumbens and ventral tegmental area. Only protein kinase A activity levels were altered by cocaine treatment, but this alteration persisted for only 7 days, whereas a sensitized locomotor response was still evident at 21 days. Although behavioral tolerance to cocaine was seen the day after the termination of treatment, none of the molecular measures was altered on this or any other day. Thus, although increased protein kinase A activity can temporarily modulate sensitized responses to cocaine, alterations in total levels of the molecules assessed in our study do not correlate with the expression of sensitized or tolerant locomotor responses to cocaine.

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.

Sexual sensation seeking, reduced concern about HIV and sexual risk behaviour among gay men in primary relationships.

Gay and bisexual men who indicated they were currently in a primary relationship with another man (N = 230) completed measures of HIV treatment attitudes, sexual risk behaviour and sexual sensation seeking. Results indicate non-primary partner sexual activity is common in many gay relationships and men in non-exclusive relationships possessed greater levels of sexual sensation seeking and treatment-related reduced concern about the dangerousness of HIV than men in exclusive relationships. Results also suggest that individuals who were members of HIV-seroconcordant relationships were more likely to engage in unprotected sexual activity with their primary sexual partners than gay men who were members of HIV-discordant couples. A series of regression analyses revealed that reduced concern about HIV mediated the relationship between sexual sensation seeking and sexual risk behaviour. The next generation of HIV prevention interventions must address the attitudinal shifts that have occurred among some gay men regarding the seriousness of HIV and should be sensitive to the dynamics of gay relationships.

Effect of cocaine and sucrose withdrawal period on extinction behavior, cue-induced reinstatement, and protein levels of the dopamine transporter and tyrosine hydroxylase in limbic and cortical areas in rats.

Lever pressing during tests for resistance to extinction and cue-induced reinstatement of cocaine seeking in rats progressively increases over the first 2 months of withdrawal. In the present report, we investigated the generality of these findings in rats trained to self-administer sucrose, a non-drug reinforcer. We also examined whether the time-dependent changes in cocaine seeking correlate with the levels of the dopamine transporter (DAT) and tyrosine hydroxylase (TH) proteins in the amygdala, nucleus accumbens, prefrontal cortex and orbitofrontal cortex. Rats were trained to self-administer cocaine (0.5 mg/kg/i.v. infusion) or 10% sucrose (0.2 ml/infusion into a liquid drop receptacle) for 10 days (6 h/day); each reward delivery was paired with a tone+light cue. Tests for cocaine seeking were conducted following 1 or 15 reward-free days. On the test day, rats were initially tested for resistance to extinction during 6-7 60-min extinction sessions in the absence of the tone-light cue, until they reached the extinction criterion of less than 15 responses/60 min. Subsequently, rats were tested for cue-induced reinstatement during a 60-min session in which each lever press led to a contingent presentation of the tone-light cue. Lever pressing during the tests for reward seeking was significantly greater on day 15 than on day 1 following withdrawal from both cocaine and sucrose self-administration training. The levels of DAT, but not TH, were greater in the prefrontal cortex of cocaine-trained rats than in sucrose-trained rats on both days 1 and 15 of withdrawal. The levels of DAT and TH in other brain areas were not altered following withdrawal from cocaine or sucrose self-administration. These data suggest that the withdrawal can modulate reward seeking of both drug and non-drug reinforcers, and that alterations in DAT and TH levels in the brain regions examined do not mediate this effect.

An overview of the Salmonella enteritidis risk assessment for shell eggs and egg products.

This article summarizes a quantitative microbial risk assessment designed to characterize the public health impact of consumption of shell eggs and egg products contaminated with Salmonella Enteritidis (SE). This risk assessment's objectives were to: (1) establish the baseline risk of foodborne illness from SE, (2) identify and evaluate potential risk mitigation strategies, and (3) identify data gaps related to future research efforts. The risk assessment model has five modules. The Egg Production module estimates the number of eggs produced that are SE-contaminated. Shell Egg Processing, Egg Products Processing, and Preparation & Consumption modules estimate the increase or decrease in the numbers of SE organisms in eggs or egg products as they pass through storage, transportation, processing, and preparation. A Public Health Outcomes module then calculates the incidence of illnesses and four clinical outcomes, as well as the cases of reactive arthritis associated with SE infection following consumption. The baseline model estimates an average production of 2.3 million SE-contaminated shell eggs/year of the estimated 69 billion produced annually and predicts an average of 661,633, human illnesses per year from consumption of these eggs. The model estimates approximately 94% of these cases recover without medical care, 5% visit a physician, an additional 0.5% are hospitalized, and 0.05% result in death. The contribution of SE from commercially pasteurized egg products was estimated to be negligible. Five mitigation scenarios were selected for comparison of their individual and combined effects on the number of human illnesses. Results suggest that mitigation in only one segment of the farm-to-table continuum will be less effective than several applied in different segments. Key data gaps and areas for future research include the epidemiology of SE on farms, the bacteriology of SE in eggs, human behavior in food handling and preparation, and human responses to SE exposure.

Expectancies of sexual "escape" and sexual risk among drug and alcohol-involved gay and bisexual men.

PURPOSE: We tested the hypotheses that sexual risk would relate to gay/bisexual men's patterns of combining alcohol or drugs with sex, their motivation to use drugs to cognitively "escape" awareness of HIV risk, and their use of bars as social and sexual settings. METHODS: We conducted extensive interviews among African-American (n = 139) and White (n = 112) gay and bisexual men who were attending a behavioral intervention for safer sex results. Those who frequently combined drugs with sex reported higher rates of sexual risk and Hepatitis B infection than did men who infrequently combined substances with sex, or who combined only alcohol with sex. Sexual risk was pronounced among more frequent drug users who also reported strong expectancies that alcohol or drugs facilitate sex and cognitively escape from awareness of HIV risk. Frequenting bars per se was not an important factor in sexual risk. IMPLICATIONS: Men who use alcohol or drugs to enhance sexuality and escape self-awareness of HIV risk have a significantly diminished capacity to avoid sexual risk.

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.