Genome-Wide Association Study of Behavioral Disinhibition in a Selected Adolescent Sample.

Behavioral disinhibition (BD) is a quantitative measure designed to capture the heritable variation encompassing risky and impulsive behaviors. As a result, BD represents an ideal target for discovering genetic loci that predispose individuals to a wide range of antisocial behaviors and substance misuse that together represent a large cost to society as a whole. Published genome-wide association studies (GWAS) have examined specific phenotypes that fall under the umbrella of BD (e.g. alcohol dependence, conduct disorder); however no GWAS has specifically examined the overall BD construct. We conducted a GWAS of BD using a sample of 1,901 adolescents over-selected for characteristics that define high BD, such as substance and antisocial behavior problems, finding no individual locus that surpassed genome-wide significance. Although no single SNP was significantly associated with BD, restricted maximum likelihood analysis estimated that 49.3 % of the variance in BD within the Caucasian sub-sample was accounted for by the genotyped SNPs (p = 0.06). Gene-based tests identified seven genes associated with BD (p ≤ 2.0 × 10(-6)). Although the current study was unable to identify specific SNPs or pathways with replicable effects on BD, the substantial sample variance that could be explained by all genotyped SNPs suggests that larger studies could successfully identify common variants associated with BD.

Brain cortical thickness in male adolescents with serious substance use and conduct problems.

BACKGROUND: Adolescents with substance use disorder (SUD) and conduct problems exhibit high levels of impulsivity and poor self-control. Limited work to date tests for brain cortical thickness differences in these youths. OBJECTIVES: To investigate differences in cortical thickness between adolescents with substance use and conduct problems and controls. METHODS: We recruited 25 male adolescents with SUD, and 19 male adolescent controls, and completed structural 3T magnetic resonance brain imaging. Using the surface-based morphometry software FreeSurfer, we completed region-of-interest (ROI) analyses for group cortical thickness differences in left, and separately right, inferior frontal gyrus (IFG), orbitofrontal cortex (OFC) and insula. Using FreeSurfer, we completed whole-cerebrum analyses of group differences in cortical thickness. RESULTS: Versus controls, the SUD group showed no cortical thickness differences in ROI analyses. Controlling for age and IQ, no regions with cortical thickness differences were found using whole-cerebrum analyses (though secondary analyses co-varying IQ and whole-cerebrum cortical thickness yielded a between-group cortical thickness difference in the left posterior cingulate/precuneus). Secondary findings showed that the SUD group, relative to controls, demonstrated significantly less right > left asymmetry in IFG, had weaker insular-to-whole-cerebrum cortical thickness correlations, and showed a positive association between conduct disorder symptom count and cortical thickness in a superior temporal gyrus cluster. CONCLUSION: Functional group differences may reflect a more nuanced cortical morphometric difference than ROI cortical thickness. Further investigation of morphometric differences is needed. If replicable findings can be established, they may aid in developing improved diagnostic or more targeted treatment approaches.

Transient nature of late Pleistocene climate variability.

Climate in the early Pleistocene varied with a period of 41 kyr and was related to variations in Earth's obliquity. About 900 kyr ago, variability increased and oscillated primarily at a period of approximately 100 kyr, suggesting that the link was then with the eccentricity of Earth's orbit. This transition has often been attributed to a nonlinear response to small changes in external boundary conditions. Here we propose that increasing variablility within the past million years may indicate that the climate system was approaching a second climate bifurcation point, after which it would transition again to a new stable state characterized by permanent mid-latitude Northern Hemisphere glaciation. From this perspective the past million years can be viewed as a transient interval in the evolution of Earth's climate. We support our hypothesis using a coupled energy-balance/ice-sheet model, which furthermore predicts that the future transition would involve a large expansion of the Eurasian ice sheet. The process responsible for the abrupt change seems to be the albedo discontinuity at the snow-ice edge. The best-fit model run, which explains almost 60% of the variance in global ice volume during the past 400 kyr, predicts a rapid transition in the geologically near future to the proposed glacial state. Should it be attained, this state would be more 'symmetric' than the present climate, with comparable areas of ice/sea-ice cover in each hemisphere, and would represent the culmination of 50 million years of evolution from bipolar nonglacial climates to bipolar glacial climates.

An overview of and rationale for changes proposed for pathological gambling in DSM-5.

The fifth revision of the Diagnostic and Statistical Manual of Mental Disorders (DSM-5) is scheduled for publication in 2013. It will include several changes to the diagnosis of pathological gambling: the name of the disorder will be altered, the threshold for diagnosis will decrease, and one criterion will be removed. This paper reviews the rationale for these changes and addresses how they may impact diagnosis and treatment of the disorder, as well as potential for future research in the field.

Prevalence and predictors of injection drug use and risky sexual behaviors among adolescents in substance treatment.

BACKGROUND AND OBJECTIVES: The longitudinal risk for human immunodeficiency virus (HIV) infection following adolescent substance treatment is not known. Therefore, it is not known if adolescent substance treatment should include HIV prevention interventions. To address this important research gap, this study evaluates the longitudinal prevalence and predictors of injection drug use (IDU) and sex risk behaviors among adolescents in substance treatment. METHODS: Participants were 260 adolescents (13-18 years) in substance treatment and 201 community control adolescents (11-19 years). Participants were assessed at baseline and follow-up (mean time between assessments = 6.9 years for the clinical sample and 5.6 years for the community control sample). Outcomes included self-report lifetime history of IDU, number of lifetime sex partners and frequency of unprotected sexual intercourse. RESULTS: At baseline, 7.5% of the clinical sample, compared to 1.0% of the community control sample had a lifetime history of IDU (χ12=10.53, p = .001). At follow-up, 17.4% of the clinical sample compared to 0% of the community control sample had a lifetime history of IDU (χ12=26.61, p = .0005). The number of baseline substance use disorders and onset age of marijuana use significantly predicted the presence of lifetime IDU at follow-up, after adjusting for baseline age, race, and sex. The clinical sample reported more lifetime sex partners and more frequent unprotected sex than the community control sample at baseline and follow-up. CONCLUSIONS: Many adolescents in substance treatment develop IDU and report persistent risky sex. Effective risk reduction interventions for adolescents in substance treatment are needed that address both IDU and risky sex.

Externalizing behaviors are associated with SNPs in the CHRNA5/CHRNA3/CHRNB4 gene cluster.

There is strong evidence for shared genetic factors contributing to childhood externalizing disorders and substance abuse. Externalizing disorders often precede early substance experimentation, leading to the idea that individuals inherit a genetic vulnerability to generalized disinhibitory psychopathology. Genetic variation in the CHRNA5/CHRNA3/CHRNB4 gene cluster has been associated with early substance experimentation, nicotine dependence, and other drug behaviors. This study examines whether the CHRNA5/CHRNA3/CHRNB4 locus is correlated also with externalizing behaviors in three independent longitudinally assessed adolescent samples. We developed a common externalizing behavior phenotype from the available measures in the three samples, and tested for association with 10 SNPs in the gene cluster. Significant results were detected in two of the samples, including rs8040868, which remained significant after controlling for smoking quantity. These results expand on previous work focused mainly on drug behaviors, and support the hypothesis that variation in the CHRNA5/CHRNA3/CHRNB4 locus is associated with early externalizing behaviors.

Climate sensitivity constrained by temperature reconstructions over the past seven centuries.

The magnitude and impact of future global warming depends on the sensitivity of the climate system to changes in greenhouse gas concentrations. The commonly accepted range for the equilibrium global mean temperature change in response to a doubling of the atmospheric carbon dioxide concentration, termed climate sensitivity, is 1.5-4.5 K (ref. 2). A number of observational studies, however, find a substantial probability of significantly higher sensitivities, yielding upper limits on climate sensitivity of 7.7 K to above 9 K (refs 3-8). Here we demonstrate that such observational estimates of climate sensitivity can be tightened if reconstructions of Northern Hemisphere temperature over the past several centuries are considered. We use large-ensemble energy balance modelling and simulate the temperature response to past solar, volcanic and greenhouse gas forcing to determine which climate sensitivities yield simulations that are in agreement with proxy reconstructions. After accounting for the uncertainty in reconstructions and estimates of past external forcing, we find an independent estimate of climate sensitivity that is very similar to those from instrumental data. If the latter are combined with the result from all proxy reconstructions, then the 5-95 per cent range shrinks to 1.5-6.2 K, thus substantially reducing the probability of very high climate sensitivity.

The neuronal nicotinic receptor subunit genes (CHRNA6 and CHRNB3) are associated with subjective responses to tobacco.

Neuronal nicotinic acetylcholine receptors have been implicated in various measures of nicotine dependence. In this paper, we present findings from an exploratory study of single nucleotide polymorphisms (SNPs) in the CHRNB3 and CHRNA6 genes with tobacco and alcohol phenotypes, including frequency of use and three subjective response factors occurring shortly after initiation of use. Subjects were 1056 ethnically diverse adolescents ascertained from clinical and community settings. The most significant associations were found between two CHRNB3 SNPs (rs4950 and rs13280604) and the three subjective response factors to initial tobacco use. These findings were replicated in a separate community sample of 1524 families participating in the National Longitudinal Study of Adolescent Health. Both CHRNB3 SNPs were found to be associated with similar measures of subjective response to tobacco. These results indicate that early subjective response to nicotine may be a valuable endophenotype for genetic studies aimed at uncovering genes contributing to nicotine use and addiction.

Global gene expression analysis of lenses from different mouse strains and in the alpha3Cx46 knockout mouse.

PURPOSE: Disruption of the mouse gene encoding the gap junction subunit alpha3 connexin 46 (alpha3Cx46) results in the formation of lens cataracts that have a severity affected by the genetic background of the mouse strain. To identify the genes that influence the severity of the nuclear opacity, global gene expression was analyzed in lenses from the 129SvJae strain and compared to the C57BL/6J strain. METHODS: Lens transcripts were subjected to cDNA microarray analysis. Results on selected genes were confirmed by real-time PCR. RESULTS: GENES THAT WERE DETERMINED TO BE ALTERED IN EXPRESSION LEVELS AS A RESULT OF STRAIN DIFFERENCES COULD BE CLUSTERED INTO THREE GROUPS: energy metabolism, stress response, and cell growth. CONCLUSIONS: There were no observed changes in gene expression as a result of the lack of alpha3Cx46 in the different mouse strains, suggesting that the pathways mediated by this connexin do not influence gene transcription in the lens. Analysis of the transcript changes due to strain differences provides new insights into potential genetic modifiers of cataractogenesis. More detailed experimentation will be needed to determine if these observed changes do indeed affect cataractogenesis.

Ventral striatal blood flow is altered by acute nicotine but not withdrawal from nicotine.

Neural mechanisms underlying the reinforcing effects of nicotine and other drugs have been widely studied and are known to involve the ventral striatum, which is part of the mesocorticolimbic dopamine system. In contrast, mechanisms of nicotine withdrawal have received less attention although subjective withdrawal likely contributes to the difficulty of quitting. The goal of this study was to determine if nicotine withdrawal was associated with alterations of cerebral blood flow (CBF) in ventral striatum. Twelve smokers, moderately dependent on nicotine, underwent MR dynamic susceptibility contrast (DSC) imaging at baseline, after overnight withdrawal from nicotine, and after nicotine replacement. DSC images were used to calculate CBF in three regions of interest: ventral striatum, thalamus, and medial frontal cortex. Subjective withdrawal symptoms were measured at each time point. In spite of significant subjective withdrawal symptoms, there was no main effect of withdrawal on CBF in the three regions. However, there was a significant correlation between the increase in withdrawal symptoms and a reduction in thalamic CBF. In contrast to withdrawal, nicotine replacement significantly increased CBF in ventral striatum. Our findings are consistent with the known role of ventral striatum in drug reward. The lack of a main effect on withdrawal, but correlation of thalamic blood flow with withdrawal symptoms suggests that more complex mechanisms mediate the subjective features of the withdrawal state.

Case-control and within-family tests for an association between conduct disorder and DAT1.

BACKGROUND: Conduct disorder (CD) is characterized by a persistent pattern of violating age-appropriate norms and the rights of others, and is one of the most frequently diagnosed disorders among children. CD is moderately heritable, but we know of no reliable associations with specific genes. Evidence suggests that a variable number tandem repeat polymorphism of the dopamine transporter (DAT1) gene may be associated with externalizing behavior in children. OBJECTIVE: To test for an association between the DAT1 gene and CD. DESIGN: Case-control analyses and a transmission disequilibrium test (TDT) were conducted. SETTING/PARTICIPANTS: Cases were (n=210) adolescents enrolled in a Colorado treatment program for conduct and substance use problems. Controls included adolescents matched to the probands in the treatment program and their siblings (n=162). The TDT was conducted using case families in which DNA from both parents was available (95 trios). RESULTS: The case-control analysis of the full sample did not result in a significant association [chi2 (2,372)=0.13, P=0.94]. Cases with early-onset conduct problems had slightly more 10-repeat alleles than controls, although this difference was not significant [chi2 (2,264)=2.19, P=0.33, 9/10 odds ratio (OR)=1.58, 10/10 OR=2.14]. The TDT also did not result in a significant association [chi2(1)=0.12, P=0.94]. CONCLUSION: Results did not support an association between this polymorphism of the DAT1 gene and CD in adolescents.

Item response theory analysis of DSM-IV cannabis abuse and dependence criteria in adolescents.

OBJECTIVE: To examine three aspects of adolescent cannabis problems: do DSM-IV cannabis abuse and dependence criteria represent two different levels of severity of substance involvement, to what degree do each of the 11 abuse and dependence criteria assess adolescent cannabis problems, and do the DSM-IV items function similarly across different adolescent populations? METHOD: We examined 5,587 adolescents ages 11 to 19, including 615 youths in treatment for substance use disorders, 179 adjudicated youths, and 4,793 youths from the community. All of the subjects were assessed with a structured diagnostic interview. Item response theory was used to analyze symptom endorsement patterns. RESULTS: Abuse and dependence criteria were not found to represent different levels of severity of problem cannabis use in any of the samples. Among the 11 abuse and dependence criteria, problems cutting down and legal problems were the least informative for distinguishing problem users. Two dependence criteria and three of the four abuse criteria indicated different severities of cannabis problems across samples. CONCLUSIONS: We found little evidence to support the idea that abuse and dependence are separate constructs for adolescent cannabis problems. Furthermore, certain abuse criteria may indicate severe substance problems, whereas specific dependence items may indicate less severe problems. The abuse items in particular need further study. These results have implications for the refinement of the current substance use disorder criteria for DSM-V.

Association of candidate genes with antisocial drug dependence in adolescents.

The Colorado Center For Antisocial Drug Dependence (CADD) is using several research designs and strategies in its study of the genetic basis for antisocial drug dependence in adolescents. This study reports single nucleotide polymorphism (SNP) association results from a targeted gene assay (SNP chip) of 231 primarily Caucasian male probands in treatment with antisocial drug dependence and a matched set of community controls. The SNP chip was designed to assay 1500 SNPs distributed across 50 candidate genes that have had associations with substance use disorders and conduct disorder. There was an average gene-wide inter-SNP interval of 3000 base pairs. After eliminating SNPs with poor signals and low minor allele frequencies, 60 nominally significant associations were found among the remaining 1073 SNPs in 18 of 49 candidate genes. Although none of the SNPs achieved genome-wide association significance levels (defined as p<.000001), two genes probed with multiple SNPs (OPRM1 and CHRNA2) emerged as plausible candidates for a role in antisocial drug dependence after gene-based permutation tests. The custom-designed SNP chip served as an effective and flexible platform for rapid interrogation of a large number of plausible candidate genes.

Brain activation during the Stroop task in adolescents with severe substance and conduct problems: A pilot study.

BACKGROUND: Although many neuroimaging studies have examined changes in brain function in adults with substance use disorders, far fewer have examined adolescents. This study investigated patterns of brain activation in adolescents with severe substance and conduct problems (SCP) compared to controls. METHODS: Functional magnetic resonance imaging (fMRI) at 1.5Tesla assessed brain activation in 12 adolescent males with SCP, ranging in age from 14 to 18, and 12 controls similar in age, gender, and neighborhood while performing the attentionally demanding Stroop task. RESULTS: Even though the adolescents with SCP performed as well as the controls, they activated a more extensive set of brain structures for incongruent (e.g., "red" in blue ink) versus congruent (e.g. "red" in red ink) trials. These regions included parahippocampal regions bilaterally, posterior regions involved in language-related processing, right-sided medial prefrontal areas, and subcortical regions including the thalamus and caudate. CONCLUSION: These preliminary results suggest that the neural mechanisms of attentional control in youth with SCP differ from youth without such problems. This difficulty may prevent SCP youth from ignoring salient but distracting information in the environment, such as drug-related information.

Test of association between TaqIA A1 allele and alcohol use disorder phenotypes in a sample of adolescent patients with serious substance and behavioral problems.

UNLABELLED: Several studies have demonstrated a significant association between the A1 allele of the TaqIA polymorphism and various phenotypes of alcoholism, others have not, and two studies have shown the reversed association, where the A2 allele was associated with higher levels of alcohol consumption. We sought to test for an association between early onset (in childhood or adolescence) alcohol use disorders and the DRD2 TaqIA polymorphism and to resolve some of the hypothesized explanations for previous negative results, utilizing a larger sample than many previous studies. METHODS: We selected individuals with a lifetime alcohol abuse or dependence (n=239) diagnosis from a clinically ascertained sample of youth (ages 13-19) with serious conduct and substance problems (about 90% also met criteria for conduct disorder and a cannabis use disorder) and compared them with individuals without a lifetime alcohol use disorder diagnosis ascertained from (1) community adolescent controls (n=151), (2) siblings of patients (n=87) and (3) other adolescent patients (n=92). Cases were compared with each control group, separately, by genotype using the chi(2)-test. Using 78 adolescent patients with an alcohol use disorder where genotypic information was available for both parents, we conducted the transmission disequilibrium test (TDT). RESULTS: Case-control results were non-significant using the entire community control sample (chi(2)(2)=1.92; p=0.38) and when restricting the sample to Caucasians (chi(2)(2)=3.81; p=0.15) or Hispanics (chi(2)(2)=1.70; p=0.43). Case-control results using the other comparison groups and TDT results were also non-significant. DISCUSSION: We did not find support for an association between the TaqIA polymorphism and early onset alcohol use disorders.

A genome-wide scan for loci influencing adolescent cannabis dependence symptoms: evidence for linkage on chromosomes 3 and 9.

OBJECTIVE: Cannabis is the most frequently abused illicit substance among adolescents and young adults. Genetic risk factors account for part of the variation in the development of cannabis dependence symptoms; however, no linkage studies have been performed for cannabis dependence symptoms. This study aimed to identify such loci. METHOD: Three hundred and twenty-four sibling pairs from 192 families were assessed for cannabis dependence symptoms. Probands (13-19 years of age) were recruited from consecutive admissions to substance abuse treatment facilities. The siblings of the probands ranged in age from 12 to 25 years. A community-based sample of 4843 adolescents and young adults was utilized to define an age- and sex-corrected index of cannabis dependence vulnerability. DSM-IV cannabis dependence symptoms were assessed in youth and their family members with the Composite International Diagnostic Instrument-Substance Abuse Module. Siblings and parents were genotyped for 374 microsatellite markers distributed across the 22 autosomes (average inter-marker distance=9.2cM). Cannabis dependence symptoms were analyzed using Merlin-regress, a regression-based method that is robust to sample selection. RESULTS: Evidence for suggestive linkage was found on chromosome 3q21 near marker D3S1267 (LOD=2.61), and on chromosome 9q34 near marker D9S1826 (LOD=2.57). CONCLUSIONS: This is the first reported linkage study of cannabis dependence symptoms. Other reports of linkage regions for illicit substance dependence have been reported near 3q21, suggesting that this region may contain a quantitative trait loci influencing cannabis dependence and other substance use disorders.

Personality risk profile for conduct disorder and substance use disorders in youth.

The five factor model of personality is a useful metric to describe personality profiles associated with maladaptive functioning. Using the NEO-Five Factor Inventory (NEO-FFI), we examined a conceptually based profile of high neuroticism, low agreeableness and low conscientiousness among 243 youth (aged 13-18 years) with varying degrees of conduct disorder (CD) and substance use disorders (SUD). Comparisons of the NEO-FFI personality dimensions between CD/SUD youth and adolescent siblings (N=173), and relations between the personality dimensions and behavioral indicators of conduct disorder and substance involvement were examined. Youth with CD and SUD had greater neuroticism, lower agreeableness, and lower conscientiousness than siblings of a similar age. The NEO-FFI scales predicted aggression and substance involvement for both probands and siblings in this cross-sectional investigation. These findings support the role for personality in models of the etiology and persistence of conduct disorder and substance use disorders.

Imaging decision about whether to benefit self by harming others: Adolescents with conduct and substance problems, with or without callous-unemotionality, or developing typically.

We sought to identify brain activation differences in conduct-problem youth with limited prosocial emotions (LPE) compared to conduct-problem youth without LPE and community adolescents, and to test associations between brain activation and severity of callous-unemotional traits. We utilized a novel task, which asks subjects to repeatedly decide whether to accept offers where they will benefit but a beneficent other will be harmed. Behavior on this task has been previously associated with levels of prosocial emotions and severity of callous-unemotional traits, and is related to empathic concern. During fMRI acquisition, 66 male adolescents (21 conduct-problem patients with LPE, 21 without, and 24 typically-developing controls) played this novel game. Within typically-developing controls, we identified a network engaged during decision involving bilateral insula, and inferior parietal and medial frontal cortices, among other regions. Group comparisons using non-parametric (distribution-free) permutation tests demonstrated LPE patients had lower activation estimates than typically-developing adolescents in right anterior insula. Additional significant group differences emerged with our a priori parametric cluster-wise inference threshold. These results suggest measurable functional brain activation differences in conduct-problem adolescents with LPE compared to typically-developing adolescents. Such differences may underscore differential treatment needs for conduct-problem males with and without LPE.

Children's brain activation during risky decision-making: A contributor to substance problems?

OBJECTIVE: Among young children excessive externalizing behaviors often predict adolescent conduct and substance use disorders. Adolescents with those disorders show aberrant brain function when choosing between risky or cautious options. We therefore asked whether similarly aberrant brain function during risky decision-making accompanies excessive externalizing behaviors among children, hypothesizing an association between externalizing severity and regional intensity of brain activation during risky decision-making. METHOD: Fifty-eight (58) 9-11 year-old children (both sexes), half community-recruited, half with substance-treated relatives, had parent-rated Child Behavior Checklist Externalizing scores. During fMRI, children repeatedly chose between doing a cautious behavior earning 1 point or a risky behavior that won 5 or lost 10 points. Conservative permutation-based whole-brain regression analyses sought brain regions where, during decision-making, activation significantly associated with externalizing score, with sex, and with their interaction. RESULTS: Before risky responses higher externalizing scores were significantly, negatively associated with neural activation (t's: 2.91-4.76) in regions including medial prefrontal cortex (monitors environmental reward-punishment schedules), insula (monitors internal motivating states, e.g., hunger, anxiety), dopaminergic striatal and midbrain structures (anticipate and mediate reward), and cerebellum (where injuries actually induce externalizing behaviors). Before cautious responses there were no significant externalizing:activation associations (except in post hoc exploratory analyses), no significant sex differences in activation, and no significant sex-by-externalizing interactions. CONCLUSIONS: Among children displaying more externalizing behaviors extensive decision-critical brain regions were hypoactive before risky behaviors. Such neural hypoactivity may contribute to the excessive real-life risky decisions that often produce externalizing behaviors. Substance exposure, minimal here, was a very unlikely cause.

Priming in response to pro-inflammatory cytokines is a feature of adult synovial but not dermal fibroblasts.

BACKGROUND: It has been hypothesized that chronic inflammatory diseases such as rheumatoid arthritis (RA) may be caused by a failure of negative feedback mechanisms. This study sought to examine negative feedback mechanisms in fibroblast-like synoviocytes (FLS), one of the most abundant cell types in the joint. We hypothesized that prior exposure of healthy FLS to an inflammatory stimulus would attenuate their responses to a second inflammatory stimulus, in the same way that negative feedback mechanisms desensitize macrophages to repeated stimulation by lipopolysaccharide. We further hypothesized that such negative feedback mechanisms would be defective in FLS derived from the joints in RA. METHODS: Synovial fibroblasts and dermal fibroblasts from non-inflamed joints and joints affected by RA and a fibroblast cell line from neonatal foreskin were stimulated twice with tumour necrosis factor (TNF) α or interleukin (IL)-1α, with a 24-h rest period between the two 24-h stimulations. Differences between response to the first and second dose of cytokine were examined by assessing secretion of inflammatory factors and intracellular signalling activity. RESULTS: FLS from both non-inflamed joints and joints affected by RA mounted an augmented response to re-stimulation. This response was site-specific, as primary dermal fibroblasts did not alter their response between doses. The fibroblast priming was also gene-specific and transient. Assessment of signalling events and nuclear localization showed prolonged activation of nuclear factor (NF)-κB during the second stimulation. CONCLUSION: This study aimed to examine mechanisms of negative regulation of inflammatory responses in FLS. Instead, we found a pro-inflammatory stromal memory in FLS obtained from both non-inflamed joints and joints affected by RA. This suggests the joint is an area at high risk of chronic inflammation, and may provide a piece in the puzzle of how chronic inflammation is established in RA.