Opioid-induced fragile-like regulatory T cells contribute to withdrawal.

Dysregulation of the immune system is a cardinal feature of opioid addiction. Here, we characterize the landscape of peripheral immune cells from patients with opioid use disorder and from healthy controls. Opioid-associated blood exhibited an abnormal distribution of immune cells characterized by a significant expansion of fragile-like regulatory T cells (Tregs), which was positively correlated with the withdrawal score. Analogously, opioid-treated mice also showed enhanced Treg-derived interferon-γ (IFN-γ) expression. IFN-γ signaling reshaped synaptic morphology in nucleus accumbens (NAc) neurons, modulating subsequent withdrawal symptoms. We demonstrate that opioids increase the expression of neuron-derived C-C motif chemokine ligand 2 (Ccl2) and disrupted blood-brain barrier (BBB) integrity through the downregulation of astrocyte-derived fatty-acid-binding protein 7 (Fabp7), which both triggered peripheral Treg infiltration into NAc. Our study demonstrates that opioids drive the expansion of fragile-like Tregs and favor peripheral Treg diapedesis across the BBB, which leads to IFN-γ-mediated synaptic instability and subsequent withdrawal symptoms.

Is melanin-concentrating hormone in the medial preoptic area a signal for the decline of maternal care in late postpartum?

This manuscript proposes that melanin-concentrating hormone (MCH) in the medial preoptic area (MPOA) is an neurochemical signal evolved to trigger the declining process of maternal care. MCH in the MPOA appears only after parturition and is progressively increased with the progression of lactation, while maternal behavior declines progressively. Intra-MPOA injection of MCH decreases active maternal responses. MCH is also highly responsive to infant characteristics and maternal condition. Behavioral changes induced by MCH in late postpartum period are conducive to the decline of infant-directed maternal behavior. The MPOA MCH system may mediate the maternal behavior decline by suppressing the maternal approach motivation and/or increasing maternal withdrawal via its inhibitory action onto the mesolimbic dopamine D1/D2 receptors and its stimulating action on serotonin 5-HT2C receptors in the ventral tegmental area. Research into the MCH maternal effects will enhance our understanding of the neurochemical mechanisms underlying the maternal behavior decline.

Impact and Role of Hypothalamic Corticotropin Releasing Hormone Neurons in Withdrawal from Chronic Alcohol Consumption in Female and Male Mice.

Worldwide, alcohol use and abuse are a leading risk of mortality, causing 5.3% of all deaths (World Health Organization, 2022). The endocrine stress system, initiated by the peripheral release of corticotropin releasing hormone (CRH) from primarily glutamatergic neurons in the paraventricular nucleus of the hypothalamus (PVN), is profoundly linked with alcohol use, abuse, and relapse (Blaine and Sinha, 2017). These PVN CRH-releasing (PVNCRH) neurons are essential for peripheral and central stress responses (Rasiah et al., 2023), but little is known about how alcohol affects these neurons. Here, we show that two-bottle choice alcohol consumption blunts the endocrine-mediated corticosterone response to stress during acute withdrawal in female mice. Conversely, using slice electrophysiology, we demonstrate that acute withdrawal engenders a hyperexcitable phenotype of PVNCRH neurons in females that is accompanied by increased glutamatergic transmission in both male and female mice. GABAergic synaptic transmission was unaffected by alcohol history. We then tested whether chemogenetic inhibition of PVNCRH neurons would restore stress response in female mice with a history of alcohol drinking in the looming disk test, which mimics an approaching predator threat. Accordingly, inhibition of PVNCRH neurons reduced active escape in hM4Di alcohol history mice only. This study indicates that stress-responsive PVNCRH neurons in females are particularly affected by a history of alcohol consumption. Interestingly, women have indicated an increase in heavy alcohol use to cope with stress (Rodriguez et al., 2020), perhaps pointing to a potential underlying mechanism in alcohol-mediated changes to PVNCRH neurons that alter stress response.SIGNIFICANCE STATEMENT Paraventricular nucleus of the hypothalamus neurons that release corticotropin releasing hormone (PVNCRH) are vital for stress response. These neurons have been understudied in relation to alcohol and withdrawal despite profound relations between stress, alcohol use disorders (AUD), and relapse. In this study, we use a variety of techniques to show that acute withdrawal from a history of alcohol impacts peripheral stress response, PVNCRH neurons, and behavior. Specifically, PVNCRH are in a hyperactive state during withdrawal, which drives an increase in active stress coping behaviors in female mice only. Understanding how alcohol use and withdrawal affects stress responding PVNCRH neurons may contribute to finding new potential targets for the treatment of alcohol use disorder.

Opioid Withdrawal Abruptly Disrupts Amygdala Circuit Function by Reducing Peptide Actions.

While the physical signs of opioid withdrawal are most readily observable, withdrawal insidiously drives relapse and contributes to compulsive drug use, by disrupting emotional learning circuits. How these circuits become disrupted during withdrawal is poorly understood. Because amygdala neurons mediate relapse, and are highly opioid sensitive, we hypothesized that opioid withdrawal would induce adaptations in these neurons, opening a window of disrupted emotional learning circuit function. Under normal physiological conditions, synaptic transmission between the basolateral amygdala (BLA) and the neighboring main island (Im) of GABAergic intercalated cells (ITCs) is strongly inhibited by endogenous opioids. Using patch-clamp electrophysiology in brain slices prepared from male rats, we reveal that opioid withdrawal abruptly reduces the ability of these peptides to inhibit neurotransmission, a direct consequence of a protein kinase A (PKA)-driven increase in the synaptic activity of peptidases. Reduced peptide control of neurotransmission in the amygdala shifts the excitatory/inhibitory balance of inputs onto accumbens-projecting amygdala cells involved in relapse. These findings provide novel insights into how peptidases control synaptic activity within the amygdala and presents restoration of endogenous peptide activity during withdrawal as a viable option to mitigate withdrawal-induced disruptions in emotional learning circuits and rescue the relapse behaviors exhibited during opioid withdrawal and beyond into abstinence.SIGNIFICANCE STATEMENT We find that opioid withdrawal dials down inhibitory neuropeptide activity in the amygdala. This disrupts both GABAergic and glutamatergic transmission through amygdala circuits, including reward-related outputs to the nucleus accumbens. This likely disrupts peptide-dependent emotional learning processes in the amygdala during withdrawal and may direct behavior toward compulsive drug use.

Differential Patterns of Synaptic Plasticity in the Nucleus Accumbens Caused by Continuous and Interrupted Morphine Exposure.

Opioid exposure and withdrawal both cause adaptations in brain circuits that may contribute to abuse liability. These adaptations vary in magnitude and direction following different patterns of opioid exposure, but few studies have systematically manipulated the pattern of opioid administration while measuring neurobiological impact. In this study, we compared cellular and synaptic adaptations in the nucleus accumbens shell caused by morphine exposure that was either continuous or interrupted by daily bouts of naloxone-precipitated withdrawal. At the behavioral level, continuous morphine administration caused psychomotor tolerance, which was reversed when the continuity of morphine action was interrupted by naloxone-precipitated withdrawal. Using ex vivo slice electrophysiology in female and male mice, we investigated how these patterns of morphine administration altered intrinsic excitability and synaptic plasticity of medium spiny neurons (MSNs) expressing the D1 or D2 dopamine receptor. We found that morphine-evoked adaptations at excitatory synapses were predominately conserved between patterns of administration, but there were divergent effects on inhibitory synapses and the subsequent balance between excitatory and inhibitory synaptic input. Overall, our data suggest that continuous morphine administration produces adaptations that dampen the output of D1-MSNs, which are canonically thought to promote reward-related behaviors. Interruption of otherwise continuous morphine exposure does not dampen D1-MSN functional output to the same extent, which may enhance behavioral responses to subsequent opioid exposure. Our findings support the hypothesis that maintaining continuity of opioid administration could be an effective therapeutic strategy to minimize the vulnerability to opioid use disorders.SIGNIFICANCE STATEMENT Withdrawal plays a key role in the cycle of addiction to opioids like morphine. We studied how repeated cycles of naloxone-precipitated withdrawal from otherwise continuous opioid exposure can change brain function of the nucleus accumbens, which is an important brain region for reward and addiction. Different patterns of opioid exposure caused unique changes in communication between neurons in the nucleus accumbens, and the nature of these changes depended on the type of neuron being studied. The specific changes in communication between neurons caused by repeated cycles of withdrawal may increase vulnerability to opioid use disorders. This highlights the importance of reducing or preventing the experience of withdrawal during opioid treatment.

Extracellular ATP Neurotransmission and Nicotine Sex-Specifically Modulate Habenular Neuronal Activity in Adolescence.

The recent increase in the use of nicotine products by teenagers has revealed an urgent need to better understand the impact of nicotine on the adolescent brain. Here, we sought to examine the actions of extracellular ATP as a neurotransmitter and to investigate whether ATP and nicotinic signaling interact during adolescence. With the GRABATP (G-protein-coupled receptor activation-based ATP sensor), we first demonstrated that nicotine induces extracellular ATP release in the medial habenula, a brain region involved in nicotine aversion and withdrawal. Using patch-clamp electrophysiology, we then demonstrated that activation of the ATP receptors P2X or P2Y1 increases the neuronal firing of cholinergic neurons. Surprisingly, contrasting interactive effects were observed with nicotine exposure. For the P2X receptor, activation had no observable effect on acute nicotine-mediated activity, but during abstinence after 10 d of nicotine exposure, coexposure to nicotine and the P2X agonist potentiated neuronal activity in female, but not male, neurons. For P2Y1 signaling, a potentiated effect of the agonist and nicotine was observed with acute exposure, but not following extended nicotine exposure. These data reveal a complex interactive effect between nicotinic and ATP signaling in the adolescent brain and provide mechanistic insights into extracellular ATP signaling with sex-specific alterations of neuronal responses based on prior drug exposure.SIGNIFICANCE STATEMENT In these studies, it was discovered that nicotine induces extracellular ATP release in the medial habenula and subsequent activation of the ATP purinergic receptors increases habenular cholinergic neuronal firing in the adolescent brain. Interestingly, following extended nicotine exposure, nicotine was found to alter the interplay between purinergic and nicotinic signaling in a sex-specific manner. Together, these studies provide a novel understanding for the role of extracellular ATP in mediating habenular activity and reveal how nicotine exposure during adolescence alters these signaling mechanisms, which has important implications given the high incidence of e-cigarette/vape use by youth.

Specific Inhibition of Interpeduncular Nucleus GABAergic Neurons Alleviates Anxiety-Like Behaviors in Male Mice after Prolonged Abstinence from Methamphetamine.

Anxiety is one of the most common withdrawal symptoms of methamphetamine (METH) abuse, which further drives relapse to drugs. Interpeduncular nucleus (IPN) has been implicated in anxiety-like behaviors and addiction, yet its role in METH-abstinence-induced anxiety remains unknown. Here, we found that prolonged abstinence from METH enhanced anxiety-like behaviors in male mice, accompanied by more excited IPN GABAergic neurons, as indicated by the increased c-fos expression and the enhanced neuronal excitability by electrophysiological recording in the GABAergic neurons. Using the designer receptors exclusively activated by designer drugs method, specific inhibition of IPN GABAergic neurons rescued the aberrant neuronal excitation of IPN GABAergic neurons and efficiently reduced anxiety-like behaviors, whereas it did not induce depression-like behaviors in male mice after prolonged abstinence from METH. These findings reveal that IPN GABAergic neurons should be a promising brain target to alleviate late withdrawal symptoms from METH with few side effects.SIGNIFICANCE STATEMENT Prolonged abstinence from METH triggers IPN GABAergic neurons and ultimately increases anxiety in male mice. Suppressing IPN GABAergic neurons rescues METH abstinence-induced aberrant neuronal excitation of IPN GABAergic neurons and efficiently reduces anxiety in mice.

Blinded Withdrawal of Long-Term Randomized Treatment With Empagliflozin or Placebo in Patients With Heart Failure.

BACKGROUND: It is not known whether the benefits of sodium-glucose cotransporter 2 inhibitors in heart failure persist after years of therapy. METHODS: In the EMPEROR-Reduced (Empagliflozin Outcome Trials in Chronic Heart Failure With Reduced Ejection Fraction) and EMPEROR-Preserved (Empagliflozin Outcome Trials in Chronic Heart Failure With Preserved Ejection Fraction) trials, patients with heart failure were randomly assigned (double-blind) to placebo or empagliflozin 10 mg/day for a median of 16 and 26 months, respectively. At the end of the trials, 6799 patients (placebo 3381, empagliflozin 3418) were prospectively withdrawn from treatment in a blinded manner, and, of these, 3981 patients (placebo 2020, empagliflozin 1961) underwent prespecified in-person assessments after ≈30 days off treatment. RESULTS: From 90 days from the start of closeout to the end of double-blind treatment, the annualized risk of cardiovascular death or hospitalization for heart failure was lower in empagliflozin-treated patients than in placebo-treated patients (10.7 [95% CI, 9.0-12.6] versus 13.5 [95% CI, 11.5-15.6] events per 100 patient-years, respectively; hazard ratio 0.76 [95% CI, 0.60-0.96]). When the study drugs were withdrawn for ≈30 days, the annualized risk of cardiovascular death or hospitalization for heart failure increased in patients withdrawn from empagliflozin but not in those withdrawn from placebo (17.0 [95% CI, 12.6-22.1] versus 14.1 [95% CI, 10.1-18.8] events per 100 patient-years for empagliflozin and placebo, respectively). The hazard ratio for the change in risk in the patients withdrawn from empagliflozin was 1.75 (95% CI, 1.20-2.54), P=0.0034, whereas the change in the risk in patients withdrawn from placebo was not significant (hazard ratio 1.12 [95% CI, 0.76-1.66]); time period-by-treatment interaction, P=0.068. After withdrawal, the Kansas City Cardiomyopathy Questionnaire Clinical Summary Score declined by 1.6±0.4 in patients withdrawn from empagliflozin versus placebo (P<0.0001). Furthermore, withdrawal of empagliflozin was accompanied by increases in fasting glucose, body weight, systolic blood pressure, estimated glomerular filtration rate, N-terminal pro-hormone B-type natriuretic peptide, uric acid, and serum bicarbonate and decreases in hemoglobin and hematocrit (all P<0.01). These physiological and laboratory changes were the inverse of the effects of the drug seen at the start of the trials during the initiation of treatment (≈1-3 years earlier) in the same cohort of patients. CONCLUSIONS: These observations demonstrate a persistent effect of empagliflozin in patients with heart failure even after years of treatment, which dissipated rapidly after withdrawal of the drug. REGISTRATION: URL: https://www. CLINICALTRIALS: gov; Unique identifiers: NCT03057977 and NCT03057951.

Differential patterns of basal and naloxone-evoked dopamine efflux in the rat dorsal and ventral striatum following prolonged-intermittent exposure to morphine.

Hypodopaminergia in the ventral striatum is a putative neurobiological correlate of withdrawal in opioid-dependent individuals. This perspective stands in contrast to brain imaging studies with chronic opioid users showing that naloxone-enhanced dopamine (DA) release in the dorsal striatum is positively correlated with withdrawal aversion. Here, we examined regional differences in striatal DA function associated with opioid withdrawal in rats exposed to intermittent morphine injections for 31 days. Basal concentrations of DA were reduced (i.e., indicating a hypodopaminergic state) in the ventral striatum on Day 10 of morphine exposure, whereas a more prolonged period of morphine treatment was required to reveal hypodopaminergia in the dorsal striatum on Day 31. The ventral striatum consistently exhibited naloxone-induced transient reductions in DA below the hypodopaminergic basal levels, whereas morphine enhanced DA efflux. In the dorsal striatum, DA responsivity to naloxone shifted from a significant decrease on Day 10 to a notable increase above hypodopaminergic basal levels on Day 31, corroborating the findings in the human dorsal striatum. Unexpectedly, the magnitude of morphine-evoked increases in DA efflux on Day 31 was significantly blunted relative to values on Day 10. These findings indicate that prolonged-intermittent access to morphine results in a sustained hypodopaminergic state as reflected in basal levels in the striatum, which is accompanied by regional differences in DA responsivity to naloxone and morphine. Overall, our findings suggest that prolonging the duration of morphine exposure to 31 days is sufficient to reveal neuroadaptations that may underlie the transition from initial drug exposure to opioid dependence.

Connexin-36-positive gap junctions in ventral tegmental area GABA neurons sustain opiate dependence.

Drug dependence is characterized by a switch in motivation wherein a positively reinforcing substance can become negatively reinforcing. Put differently, drug use can transform from a form of pleasure-seeking to a form of relief-seeking. Ventral tegmental area (VTA) GABA neurons form an anatomical point of divergence between two double dissociable pathways that have been shown to be functionally implicated and necessary for these respective motivations to seek drugs. The tegmental pedunculopontine nucleus (TPP) is necessary for opiate conditioned place preferences (CPP) in previously drug-naïve rats and mice, whereas dopaminergic (DA) transmission in the nucleus accumbens (NAc) is necessary for opiate CPP in opiate-dependent and withdrawn (ODW) rats and mice. Here, we show that this switch in functional anatomy is contingent upon the gap junction-forming protein, connexin-36 (Cx36), in VTA GABA neurons. Intra-VTA infusions of the Cx36 blocker, mefloquine, in ODW rats resulted in a reversion to a drug-naïve-like state wherein the TPP was necessary for opiate CPP and where opiate withdrawal aversions were lost. Consistent with these data, conditional knockout mice lacking Cx36 in GABA neurons (GAD65-Cre;Cx36 fl(CFP)/fl(CFP)) exhibited a perpetual drug-naïve-like state wherein opiate CPP was always DA independent, and opiate withdrawal aversions were absent even in mice subjected to an opiate dependence and withdrawal induction protocol. Further, viral-mediated rescue of Cx36 in VTA GABA neurons was sufficient to restore their susceptibility to an ODW state wherein opiate CPP was DA dependent. Our findings reveal a functional role for VTA gap junctions that has eluded prevailing circuit models of addiction.

Efficacy of transdermal buprenorphine patch for managing withdrawal symptoms in patients with cancer physically dependent on prescription opioids.

BACKGROUND: The physical dependence on prescription opioids among cancer survivors remains an under-investigated area, with a scarcity of well-designed prospective studies. METHODS: This single-arm, phase-2 clinical trial in Korea assessed the efficacy and safety of a transdermal buprenorphine patch (TBP) in managing physical dependence on prescription opioids in cancer survivors, as confirmed through the DSM-5 criteria or psychiatric consultation for opioid withdrawal. This study involved a 4-phase treatment protocol of screening, induction/stabilization, discontinuation, and monitoring. The primary outcome was the rate of successful opioid discontinuation, as measured by a negative urine-drug screening at 8 weeks. Key secondary outcomes included the resumption of prescribed opioids, changes in both the Clinical Opioid Withdrawal Scale (COWS) and morphine equivalent daily dose (MEDD), and assessments related to the psychological and physiological aspects of dependence and safety. RESULTS: Thirty-one participants were enrolled. In the intention-to-treat population, the success rate of opioid discontinuation was 58%, with only 2 participants experiencing a resumption of prescribed opioids. Significant reductions were observed in MEDD, which decreased from 98 to 26 mg/day (P < .001), and COWS scores, which decreased from 5.5 to 2.8 (P < .001). Desire to use opioids reduced from 7.0 to 3.0 on a 10-point numeric rating scale (P < .001). Toxicities related to TBP were mild and manageable, without severe precipitated withdrawal symptoms. CONCLUSION: TBP may be considered as an alternative therapeutic option in cancer survivors physically dependent on prescription opioids, especially where sublingual formulations are unavailable.

Effects of Thiopurine Withdrawal on Vedolizumab-Treated Patients With Ulcerative Colitis: A Randomized Controlled Trial.

BACKGROUND & AIMS: The impact of thiopurine de-escalation while on vedolizumab versus continuing thiopurine therapy in ulcerative colitis (UC) is unclear. We aimed to determine the effect of thiopurine withdrawal for patients with UC in remission on vedolizumab. METHODS: This multicenter randomized controlled trial recruited UC patients on vedolizumab 300 mg intravenously every 8 weeks and a thiopurine. Patients in steroid-free clinical remission for ≥6 months and endoscopic remission/improvement (Mayo endoscopic subscore ≤1) were randomized 2:1 to withdraw or continue thiopurine. Primary outcome was comparing week 48 vedolizumab trough concentrations. Secondary outcomes were clinical relapse (partial Mayo score ≥3 and fecal calprotectin >150 µg/g or increase in Mayo endoscopic subscore ≥1 from baseline), fecal calprotectin remission (<150 µg/g), C-reactive protein remission (<5 mg/L), centrally read endoscopic remission (Mayo endoscopic subscore = 0), histologic remission (Nancy index = 0), histo-endoscopic remission, and adverse events. RESULTS: In total, 62 patients were randomized to continue (n = 20) or withdraw (n = 42) thiopurine. At week 48, vedolizumab trough concentrations were not significantly different between continue and withdrawal groups (14.7 µg/mL, interquartile rate [IQR], 12.3-18.5 µg/mL versus 15.9 µg/mL, IQR, 10.1-22.7 µg/mL, respectively, P = 0.36). The continue group had significantly higher fecal calprotectin remission (95.0%, 19/20 versus 71.4%, 30/42; P = .03), histologic remission (80.0%, 16/20 versus 48.6%, 18/37; P = .02), and histo-endoscopic remission (75.0%, 15/20 versus 32.4%, 12/37; P = .002) than the withdrawal group. Histologic activity (hazard ratio [HR], 15.5; 95% confidence interval [CI], 1.6-146.5; P = .02) and prior anti-tumor necrosis factor exposure (HR, 6.5; 95% CI, 1.3-33.8; P = .03) predicted clinical relapse after thiopurine withdrawal. CONCLUSIONS: Thiopurine withdrawal did not affect vedolizumab trough concentrations. However, it may increase fecal calprotectin, histologic, and histo-endoscopic activity. Histologic activity and prior anti-tumor necrosis factor exposure may predict disease relapse on thiopurine withdrawal for patients using vedolizumab for UC. Australian and New Zealand Trial Registry, number ACTRN12618000812291.

Formation of chronic morphine withdrawal memories requires C1QL3-mediated regulation of PSD95 in the mouse basolateral amygdala.

Chronic morphine withdrawal memory formation is a complex process influenced by various molecular mechanisms. In this study, we aimed to investigate the contributions of the basolateral amygdala (BLA) and complement component 1, q subcomponent-like 3 (C1QL3), a secreted and presynaptically targeted protein, to the formation of chronic morphine (repeat dosing of morphine) withdrawal memory using conditioned place aversion (CPA) and chemogenetic methods. We conducted experiments involving the inhibition of the BLA during naloxone-induced withdrawal to assess its impact on CPA scores, providing insights into the significance of the BLA in the chronic morphine memory formation process. We also examined changes in C1ql3/C1QL3 expression within the BLA following conditioning. Immunofluorescence analysis revealed the colocalization of C1QL3 and the G protein-coupled receptor, brain-specific angiogenesis inhibitor 3 (BAI3) in the BLA, supporting their involvement in synaptic development. Moreover, we downregulated C1QL3 expression in the BLA to investigate its role in chronic morphine withdrawal memory formation. Our findings revealed that BLA inhibition during naloxone-induced withdrawal led to a significant reduction in CPA scores, confirming the critical role of the BLA in this memory process. Additionally, the upregulation of C1ql3 expression within the BLA postconditioning suggested its participation in withdrawal memory formation. The colocalization of C1QL3 and BAI3 in the BLA further supported their involvement in synaptic development. Furthermore, downregulation of C1QL3 in the BLA effectively hindered chronic morphine withdrawal memory formation, emphasizing its pivotal role in this process. Notably, we identified postsynaptic density protein 95 (PSD95) as a potential downstream effector of C1QL3 during chronic morphine withdrawal memory formation. Blocking PSD95 led to a significant reduction in the CPA score, and it appeared that C1QL3 modulated the ubiquitination-mediated degradation of PSD95, resulting in decreased PSD95 protein levels. This study underscores the importance of the BLA, C1QL3 and PSD95 in chronic morphine withdrawal memory formation. It provides valuable insights into the underlying molecular mechanisms, emphasizing their significance in this intricate process.

Long-term effects of aromatase inhibitor withdrawal on bone mineral density in early breast cancer patients: 10-year follow-up results of the BREX study.

PURPOSE: We aimed to provide long-term bone mineral density (BMD) data on early breast cancer patients of the BREX (Breast Cancer and Exercise) study. The effects of exercise and adjuvant endocrine treatment 10 years after randomization were analyzed, with special emphasis on aromatase inhibitor (AI) therapy discontinuation at 5 years. METHODS: The BREX study randomized 573 pre- and postmenopausal breast cancer patients into a 1-year supervised exercise program or a control group. 372 patients were included into the current follow-up analysis. BMD (g/cm2) was measured by dual-energy X-ray absorptiometry at lumbar spine (LS), left femoral neck (FN), and the total hip. Separate groups were displayed according to baseline menopausal status, and whether the patient had discontinued AI therapy at 5 years or not. RESULTS: The BMD change from 5 to 10 years did not significantly differ between the two randomized arms. AI discontinuation at 5 years had statistically significant BMD effects. The FN BMD continued to decrease in patients who discontinued AI therapy during the first 5-year off-treatment, but the decrease was three-fold less than in patients without AI withdrawal (- 1.4% v. - 3.8%). The LS BMD increased (+ 2.6%) in patients with AI withdrawal during the first 5 years following treatment discontinuation, while a BMD decrease (-1.3%) was seen in patients without AI withdrawal. CONCLUSION: This study is to our knowledge the first to quantify the long-term impact of AI withdrawal on BMD. Bone loss associated with AI therapy seems partially reversible after stopping treatment. TRIAL REGISTRATION: http://www. CLINICALTRIALS: gov/ (Identifier Number NCT00639210).

YTHDF1 in periaqueductal gray inhibitory neurons contributes to morphine withdrawal responses in mice.

BACKGROUND: Physical symptoms and aversion induced by opioid withdrawal strongly affect the management of opioid addiction. YTH N6-methyladenosine (m6A) RNA binding protein 1 (YTHDF1), an m6A-binding protein, from the periaqueductal gray (PAG) reportedly contributes to morphine tolerance and hyperalgesia. However, the role of YTHDF1 in morphine withdrawal remains unclear. METHODS: A naloxone-precipitated morphine withdrawal model was established in C57/BL6 mice or transgenic mice. YTHDF1 was knocked down via adeno-associated virus transfection. Combined with the results of the single-cell RNA sequencing analysis, the changes in morphine withdrawal somatic signs and conditioned place aversion (CPA) scores were compared when YTHDF1 originating from different neurons in the ventrolateral periaqueductal gray (vlPAG) was knocked down. We further explored the role of inflammatory factors and transcription factors related to inflammatory response in morphine withdrawal. RESULTS: Our results revealed that YTHDF1 expression was upregulated in the vlPAG of mice with morphine withdrawal and that the knockdown of vlPAG YTHDF1 attenuated morphine withdrawal-related somatic signs and aversion. The levels of NF-κB and p-NF-κB were reduced after the inhibition of YTHDF1 in the vlPAG. YTHDF1 from vlPAG inhibitory neurons, rather than excitatory neurons, facilitated morphine withdrawal responses. The inhibition of YTHDF1 in vlPAG somatostatin (Sst)-expressing neurons relieved somatic signs of morphine withdrawal and aversion, whereas the knockdown of YTHDF1 in cholecystokinin (Cck)-expressing or parvalbumin (PV)-expressing neurons did not change morphine withdrawal-induced responses. The activity of c-fos + neurons, the intensity of the calcium signal, the density of dendritic spines, and the frequency of mIPSCs in the vlPAG, which were increased in mice with morphine withdrawal, were decreased with the inhibition of YTHDF1 from vlPAG inhibitory neurons or Sst-expressing neurons. Knockdown of NF-κB in Sst-expressing neurons also alleviated morphine withdrawal-induced responses. CONCLUSIONS: YTHDF1 originating from Sst-expressing neurons in the vlPAG is crucial for the modulation of morphine withdrawal responses, and the underlying mechanism might be related to the regulation of the expression and phosphorylation of NF-κB.

Withdrawal from 3-Fluoroethamphetamine induces hyperactivity and depression-like behaviors in male mice.

3-Fluoroethamphetamine (3-FEA) belongs to the amphetamine class of stimulant drugs and functions as a releasing agent for the monoamine neurotransmitters norepinephrine, dopamine, and serotonin. 3-FEA acts on the central nervous system and elicits physical and mental side effects, such as euphoria, increased heart rate, and excitement. However, little is known about the withdrawal symptoms and behavioral changes induced by 3-FEA administration. This study aimed to evaluate the short-term consequences of 3-FEA administration (twice a day, 7 days, i.p.; 1 and 10 mg/kg) in C57BL/6J mice (male, 7 weeks old) at three behavioral levels following 1-4 days of withdrawal. The evaluation included (1) withdrawal score, (2) hyperactivity (open field [OF], elevated plus maze [EPM], and cliff avoidance [CA] test), and (3) depression-like behavior (forced-swim test). In the withdrawal score test, withdrawal behavior increased in all 3-FEA groups at 16 and 40 h after withdrawal. In the OF, EPM, and CA tests, the 3-FEA administration group showed significant changes in terms of hyperactivity. In addition, in the forced-swim test, both the 1 mg/kg and 10 mg/kg 3-FEA groups showed increased immobility time. These findings indicate that 3-FEA administration may lead to physical dependence, demonstrated by the withdrawal score increase and significant changes in hyperactivity and depression-like behavior following repeated administration and drug cessation. In conclusion, this study reveals the adverse consequences of 3-FEA administration and highlights the need for awareness raising and regulatory action to control the use of this new psychoactive substance.

Immunosuppression-Free Life after Pediatric Liver Transplant: A Case-Control Study from the Society of Pediatric Liver Transplant (SPLIT) Registry.

OBJECTIVE: To compare long-term outcomes of pediatric liver transplant (LT) recipients off immunosuppression (IS) with matched controls on IS using data from the Society of Pediatric Liver Transplant (SPLIT) registry. STUDY DESIGN: This was a retrospective case-control study. SPLIT participants <18 years of age, ≥4 years after isolated LT, and off IS for ≥1 year (cases) were age- and sex-matched 1:2 to patients with the same primary diagnosis and post-LT follow-up duration (controls). Primary outcomes included retransplantation, allograft rejection, IS comorbidities, and prevalence of SPLIT-derived composite ideal outcome (c-IO) achieved at the end of the follow-up period. Differences were compared using multiple linear regression for continuous outcomes and logistic regression for dichotomous data. RESULTS: The study cohort was composed of 33 cases (42.4% male, 60.6% biliary atresia, median age at LT of 0.7 [P25, P75, 0.5, 1.6] years, median IS withdrawal time of 9 [P25, P75, 6, 12] years after LT) and 66 age- and sex-matched controls. No cases required retransplantation. Cases and controls had similar growth parameters, laboratory values, calculated glomerular filtration rates, rates of post-transplant lymphoproliferative disease, graft rejection, and attainment of c-IO. CONCLUSIONS: No differences in allograft rejection rates, IS complications, or c-IO prevalence were seen between SPLIT patients off IS and age- and sex-matched controls remaining on IS. Discontinuation of IS most commonly occurred in the context of rigorously designed IS withdrawal trials. The available sample size was small, affecting generalizability to the broader pediatric LT population.

Quantifying hospital-associated costs, and accompanying travel costs and productivity losses, before and after withdrawing TNF-α inhibitors in juvenile idiopathic arthritis.

OBJECTIVE: To quantify differences in hospital-associated costs, and accompanying travel costs and productivity losses, before and after withdrawing TNF-α inhibitors (TNFi) in JIA patients. METHODS: This was a retrospective analysis of prospectively collected data from electronic medical records of paediatric JIA patients treated with TNFi, which were immediately discontinued, spaced (increased treatment interval) or tapered (reduced subsequent doses). Costs of hospital-associated resource use (consultations, medication, radiology procedures, laboratory testing, procedures under general anaesthesia, hospitalization) and associated travel costs and productivity losses were quantified during clinically inactive disease until TNFi withdrawal (pre-withdrawal period) and compared with costs during the first and second year after withdrawal initiation (first and second year post-withdrawal). RESULTS: Fifty-six patients were included of whom 26 immediately discontinued TNFi, 30 spaced and zero tapered. Mean annual costs were €9165/patient on active treatment (pre-withdrawal) and decreased significantly to €5063/patient (-44.8%) and €6569/patient (-28.3%) in the first and second year post-withdrawal, respectively (P < 0.05). Of these total annual costs, travel costs plus productivity losses were €834/patient, €1180/patient, and €1320/patient in the three periods respectively. Medication comprised 80.7%, 61.5% and 72.4% of total annual costs in the pre-withdrawal, first and second year post-withdrawal period, respectively. CONCLUSION: In the first two years after initiating withdrawal, the total annual costs were decreased compared with the pre-withdrawal period. However, cost reductions were lower in the second year compared with the first year post-withdrawal, primarily due to restarting or intensifying biologics. To support biologic withdrawal decisions, future research should assess the full long-term societal cost impacts, and include all biologics.

Tocilizumab discontinuation after remission achievement in patients with adult-onset Still's disease.

OBJECTIVES: Tocilizumab, an IL-6 inhibitor, has been proven effective in patients with adult-onset Still's disease (AOSD). This study aimed to clarify whether tocilizumab can be discontinued after achieving remission and to identify factors relevant to its successful discontinuation. METHODS: Consecutive patients with AOSD diagnosed according to Yamaguchi's criteria from April 2012 to July 2022, who were treated with tocilizumab, were retrospectively reviewed. RESULTS: Forty-eight patients with AOSD treated with intravenous tocilizumab, with sufficient information, were included. Thirty-eight patients (79.2%) achieved remission after 6 months of tocilizumab treatment, 12 of whom discontinued tocilizumab during remission. Within 1 year after tocilizumab discontinuation, six patients (50.0%) recurred at a mean of 5.5 months, while the other six (50.0%) remained in remission. Between the non-recurrence and recurrence groups, no difference was found in disease activity at tocilizumab discontinuation (systemic feature score, p = 0.24; ferritin, p = 0.46). While the duration of tocilizumab use was not different (p = 0.32), the interval of tocilizumab administration at tocilizumab discontinuation in the recurrence group was 21 (14-35) days, which tended to be shorter than 35 (28-53) days in the non-recurrence group (p = 0.08). Patients with prednisolone dose < 7 mg/day at last tocilizumab treatment had fewer recurrences than those without (p = 0.001). After recurrence, tocilizumab was resumed in half of the patients, resulting in successful disease control. CONCLUSIONS: The recurrence rate after tocilizumab discontinuation was 50% in 1 year. Patients who remained in remission with a longer interval of tocilizumab administration and lower prednisolone dose were likely to succeed in the withdrawal of tocilizumab.

Possibility of maintaining remission with topical therapy alone after withdrawal of dupilumab in Japanese patients with atopic dermatitis and their characteristics in the real world.

Psossibility and appropriate timing of discontinuation of dupilumab for atopic dermatitis (AD) remain unclear. We explored the possibility of patients, who could maintain remission with topical therapy alone after withdrawing dupilumab in the real world. Furthermore, we identified their characteristics. All adult AD patients who initiated dupilumab from June 2018 to July 2022 and were treated with dupilumab for more than 3 months at our hospital were included in this study. The observation period was from June 2018 to July 2023. In 138 patients, 58 (42.0%) discontinued dupilumab at least once. Among them, 18 (13.0%) discontinued dupilumab but reinitiated dupilumab later due to exacerbation. Only seven patients (5.1%) could maintain remission with topical therapy alone after discontinuation of dupilumab, with characteristics of lower POEM, VAS of pruritus, serum levels of TARC and LDH, and neutrophil counts at baseline, and those of longer duration of dupilumab until its discontinuation (24.0 ± 13.3 vs. 12.8 ± 7.3 months) and lower EASI and affected BSA at the discontinuation of dupilumab. In 118 patients treated with dupilumab for at least 1 year, 38 patients (32.2%) discontinued at least once. Only four patients (3.4%) could maintain remission with topical therapy alone after discontinuation of dupilumab, with characteristics of lower POEM at baseline and lower EASI at the discontinuation of dupilumab. In conclusion, maintaining remission after withdrawing dupilumab is challenging. Discontinuation of dupilumab may be considered in patients with low baseline POEM, after more than 2 years of dupilumab treatment, with a substantial decrease in EASI.