Cell shape sensing licenses dendritic cells for homeostatic migration to lymph nodes.

Immune cells experience large cell shape changes during environmental patrolling because of the physical constraints that they encounter while migrating through tissues. These cells can adapt to such deformation events using dedicated shape-sensing pathways. However, how shape sensing affects immune cell function is mostly unknown. Here, we identify a shape-sensing mechanism that increases the expression of the chemokine receptor CCR7 and guides dendritic cell migration from peripheral tissues to lymph nodes at steady state. This mechanism relies on the lipid metabolism enzyme cPLA2, requires nuclear envelope tensioning and is finely tuned by the ARP2/3 actin nucleation complex. We also show that this shape-sensing axis reprograms dendritic cell transcription by activating an IKKß-NF-κB-dependent pathway known to control their tolerogenic potential. These results indicate that cell shape changes experienced by immune cells can define their migratory behavior and immunoregulatory properties and reveal a contribution of the physical properties of tissues to adaptive immunity.

Traumatic Stress-Induced Increases in Anxiety-like Behavior and Alcohol Self-Administration Are Mediated by Central Amygdala CRF1 Neurons That Project to the Lateral Hypothalamus.

Avoidance stress coping, defined as persistent internal and/or external avoidance of stress-related stimuli, is a key feature of anxiety- and stress-related disorders, and contributes to increases in alcohol misuse after stress exposure. Previous work using a rat model of predator odor stress avoidance identified corticotropin-releasing factor (CRF) signaling via CRF Type 1 receptors (CRF1) in the CeA, as well as CeA projections to the lateral hypothalamus (LH) as key mediators of conditioned avoidance of stress-paired contexts and/or increased alcohol drinking after stress. Here, we report that CRF1-expressing CeA cells that project to the LH are preferentially activated in male and female rats that show persistent avoidance of predator odor stress-paired contexts (termed Avoider rats), and that chemogenetic inhibition of these cells rescues stress-induced increases in anxiety-like behavior and alcohol self-administration in male and female Avoider rats. Using slice electrophysiology, we found that prior predator odor stress exposure blunts inhibitory synaptic transmission and increases synaptic drive in CRF1 CeA-LH cells. In addition, we found that CRF bath application reduces synaptic drive in CRF1 CeA-LH cells in Non-Avoiders only. Collectively, these data show that CRF1 CeA-LH cells contribute to stress-induced increases in anxiety-like behavior and alcohol self-administration in male and female Avoider rats.SIGNIFICANCE STATEMENT Stress may lead to a variety of behavioral and physiological negative consequences, and better understanding of the neurobiological mechanisms that contribute to negative stress effects may lead to improved prevention and treatment strategies. This study, performed in laboratory rats, shows that animals that exhibit avoidance stress coping go on to develop heightened anxiety-like behavior and alcohol self-administration, and that these behaviors can be rescued by inhibiting the activity of a specific population of neurons in the central amygdala. This study also describes stress-induced physiological changes in these neurons that may contribute to their role in promoting increased anxiety and alcohol self-administration.

Effectiveness of intermittent fasting for weight loss in individuals with obesity: A meta-analysis of randomized controlled trials.

AIM: To assess whether intermittent fasting (IF) diets are associated with improvement in weight loss, metabolic parameters, and subjective well-being, in people with obesity. DATA SYNTHESIS: We performed a Meta-analysis of Randomized Controlled Trials longer than 2 months, retrieved through an extensive search on MedLine, Cochrane CENTRAL Library, and Embase online databases, comparing weight loss with IF diets and control diets in people with Body Mass index (BMI) > 30 kg/m2. We retrieved 9 trials, enrolling 540 patients. IF was not associated with a significantly greater reduction of body weight or BMI at any time point with respect to controls or in respect to continuous restricted diets, with low-to moderate quality of evidence; no significant difference in efficacy between alternate day fasting and time restricted eating was found. Differences in fasting plasma glucose, total or high-density lipoprotein cholesterol or blood pressure at any time point were not statistically significant, whereas a reduction of low-density lipoprotein cholesterol (MD -8.39 [-15.96, -0.81] mg/dl, P = 0.03; I2 = 0%) was observed at 2-4 months, but not in the longer term. Data on psychological parameters and overall well-being were insufficient to perform a formal meta-analysis, whereas a qualitative synthesis did not show any difference between IF and controls. CONCLUSIONS: IF is not associated with greater or lesser weight loss than non-intermittent fasting diets. Further data on psychological parameters and overall well-being are needed to properly assess the role of IF diets in the management of obesity.

The association between sport type and eating/body image concerns in high school students: a cross-sectional observational study.

PURPOSE: Disordered eating and body image concerns are increasingly common among adolescents, possibly representing the underpinning of eating disorders (EDs). This cross-sectional observational study aimed at investigating the relationship between various patterns of sports involvement or inactivity, and the abovementioned psychopathological dimensions. METHODS: All adolescents attending their 3rd-5th Italian grade in a single high school reported their sociodemographic and anthropometric data, their weekly sports involvement, and filled the Eating Disorders Examination Questionnaire 6.0 (EDE-Q), the Body Uneasiness Test, and the Muscle Dysmorphia Disorder Inventory (for boys). Comparisons were performed considering sex, weekly hours of activity, and different sports type (none, individual, or team sports). RESULTS: Of 744 enrolled students, 522 (70.2%) completed the survey. Girls showed higher underweight rates, preference for inactivity or individual sports, and higher psychometric scores compared to boys. Among girls, no differences were found based on time spent exercising or sports type. Inactive boys displayed worse weight- and shape-based psychopathology, higher body uneasiness, and higher appearance intolerance compared to those who devoted more time to exercise. Among boys, individual and team sports were associated with lower EDE-Q scores compared to inactivity, whereas body uneasiness and appearance intolerance were lower only in team sports. CONCLUSIONS: The study confirms the presence of remarkable sex differences in eating and body concerns of adolescents. Among boys, sports involvement is tied to lower ED psychopathology, and preference for team sports may be associated with reduced concerns. Wider longitudinal studies on will clarify the direction and specificity of these findings. LEVEL OF EVIDENCE: Level V-Cross-sectional observational study.

Effectiveness of low-carbohydrate diets for long-term weight loss in obese individuals: A meta-analysis of randomized controlled trials.

AIM: To assess whether low-carbohydrate (LC) diets are associated with differences in weight loss and well-being in people with obesity, and their cardiovascular and renal safety. MATERIALS AND METHODS: A meta-analysis of randomized controlled trials longer than 3 months, retrieved through an extensive search on MedLine and Embase databases, comparing weight loss with LC and control diets in people with body mass index (BMI) greater than 30 kg/m2 , was conducted. RESULTS: We retrieved 25 trials. Compared with controls, LC diets were associated with significant reduction of body weight at 3-4 (MD -2.59 [-3.93, -1.25] kg) and 6-8 months (MD -2.64 [-4.32, -0.95]), but no difference at 10-14 and 18-30 months, and significantly greater BMI reduction at 3-4 months (-1.66 [-2.70, -0.61] kg/m2 ), but not at other time points. Because only four trials reported data on renal function and psychological variables, renal safety and impact on well-being could not be assessed. Differences in fasting plasma glucose at any time point were not statistically significant. No significant differences in total or LDL cholesterol or blood pressure were found in the long term, whereas a long-term reduction of triglycerides (23.26 [-45.53, -0.98] mg/dl at 18-30 months), and increase of HDL cholesterol (MD 4.94 [0.30, 9.57] mg/dl at 18-30 months), were observed. CONCLUSION: LC diets are associated with greater short-term weight loss than non-carbohydrate-restricted diets and a longer term favourable effect on cardiovascular risk factors. Further evidence on long-term efficacy and renal safety is needed before LC diets can be recommended as the preferred diets in obese people.

Association between carotid artery dissection and vascular tortuosity: a case-control study.

PURPOSE: We aimed to verify if vascular tortuosity (VT) may represent a risk factor for spontaneous epiaortic vessel dissection (sEVD) in young adult patients. METHODS: We identified 304 patients aged under 55 years consecutively admitted for acute cerebrovascular events to our Stroke Unit. After checking the possibility to perform a 3D reconstruction of epiaortic vessels on CT-angiography images, we selected and compared fifty patients with sEVD (cases) with fifty-one patients without dissection (controls). VT of carotid and vertebral arteries was measured on reconstructions evaluating the vascular tortuosity index (VTI), calculated according to a specific algorithm, and the presence of kinking and coiling. Differences between groups were analyzed by Student-t test for numeric variables and chi-square test for categoric ones. A ROC curve analysis was used to look for a VTI threshold value beyond which the risk of dissection was significantly increased. RESULTS: VTI was significantly higher in cases than in controls only considering carotid arteries (p = 0.029); cases did not have a significantly higher rate of kinking and coiling than controls (p = 0.059 and 0.077, respectively). We have found a significant VTI threshold value of 27.9% (under curve area = 61.6%, p = 0.04) only for carotid artery dissection. CONCLUSION: VT appears to be associated with an increased risk of dissection for the carotid district but not for the vertebral one. The different structure, embryogenesis, and pathophysiology of dissection between the two districts could explain this finding. VTI threshold as carotid artery dissection predictor deserves confirmation in larger studies.

Relation between the size of patent foramen ovale and the volume of acute cerebral ischemic lesion in young patients with cryptogenic ischemic stroke.

BACKGROUND: Patent foramen ovale (PFO) closure is superior to medical therapy alone to prevent stroke recurrence in selected patients. Small cortical infarcts and large right to left shunts seem to identify patients who will benefit most from closure. We aimed to study the correlation between the size of the PFO and the volume of cerebral ischemic lesions in young patients with cryptogenic ischemic stroke. METHODS: PFO dimensions and acute ischemic lesion volume of 20 patients, aged<55 years, were analyzed with transesophageal echocardiography and brain magnetic resonance imaging, respectively. The association between the volume of ischemic lesions with the length of PFO, maximum separation between septum primum and septum secundum, and the combination of the twos was explored. RESULTS: A direct statistically significant correlation was found between cerebral lesion volume and maximum separation of septum primum and septum secundum (p=0.047). Length of PFO showed a non-significant trend towards an inverse correlation with lesion volume (p=0.603). Multiple linear regression analysis showed that cerebral lesion volume was dependent directly on maximum separation and inversely on length of PFO (regression coeff. -0,837; p= 0.057; 2,536, p=0.006, respectively). CONCLUSIONS: These data suggest that even small PFO might be pathogenetic in case of small cerebral infarcts and that large cerebral infarcts might be PFO related if the shunt is large. If confirmed, the combination of detailed characteristics of PFO with the volume of cerebral infarct could be integrated in a new score to select patients who would take real advantage from a percutaneous closure.

Glucocorticoid Receptor Antagonist Mifepristone Does Not Alter Innate Anxiety-Like Behavior in Genetically-Selected Marchigian Sardinian (msP) Rats.

Marchigian Sardinian alcohol-preferring (msP) rats serve as a unique model of heightened alcohol preference and anxiety disorders. Their innate enhanced stress and poor stress-coping strategies are driven by a genetic polymorphism of the corticotropin-releasing factor receptor 1 (CRF1) in brain areas involved in glucocorticoid signaling. The activation of glucocorticoid receptors (GRs) regulates the stress response, making GRs a candidate target to treat stress and anxiety. Here, we examined whether mifepristone, a GR antagonist known to reduce alcohol drinking in dependent rats, decreases innate symptoms of anxiety in msPs. Male and female msPs were compared to non-selected Wistar counterparts across three separate behavioral tests. We assessed anxiety-like behavior via the novelty-induced hypophagia (NIH) assay. Since sleep disturbances and hyperarousal are common features of stress-related disorders, we measured sleeping patterns using the comprehensive lab monitoring system (CLAMS) and stress sensitivity using acoustic startle measures. Rats received an acute administration of vehicle or mifepristone (60 mg/kg) 90 min prior to testing on NIH, acoustic startle response, and CLAMS. Our results revealed that both male and female msPs display greater anxiety-like behaviors as well as enhanced acoustic startle responses compared to Wistar counterparts. Male msPs also displayed reduced sleeping bout duration versus Wistars, and female msPs displayed greater acoustic startle responses versus male msPs. Importantly, the enhanced anxiety-like behavior and startle responses were not reduced by mifepristone. Together, these findings suggest that increased expression of stress-related behaviors in msPs are not solely mediated by acute activation of GRs.

Effect of Glucocorticoid Receptor Antagonism on Alcohol Self-Administration in Genetically-Selected Marchigian Sardinian Alcohol-Preferring and Non-Preferring Wistar Rats.

Alcoholism is a chronically relapsing disorder characterized by high alcohol intake and a negative emotional state during abstinence, which contributes to excessive drinking and susceptibility to relapse. Stress, dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis and alterations in glucocorticoid receptor (GR) function have been linked to transition from recreational consumption to alcohol use disorder (AUD). Here, we investigated the effect of pharmacological antagonisms of GR on alcohol self-administration (SA) using male and female Wistar and Marchigian Sardinian alcohol-preferring (msP) rats, a rodent line genetically selected for excessive alcohol drinking and highly sensitive to stress. Animals were trained to self-administer 10% (v/v) alcohol. Once a stable alcohol SA baseline was reached, we tested the effect of the GR antagonists mifepristone (0.0, 10, 30 and 60 mg/kg; i.p.) and CORT113176 (0.0, 10, 30 and 60 mg/kg) on alcohol SA. To evaluate whether the effects of the two compounds were specific for alcohol, the two drugs were tested on a similar saccharin SA regimen. Finally, basal blood corticosterone (CORT) levels before and after alcohol SA were determined. Systemic injection with mifepristone dose-dependently reduced alcohol SA in male and female Wistars but not in msPs. Administration of CORT113176 decreased alcohol SA in male and female Wistars as well as in female msPs but not in male msP rats. At the highest dose, mifepristone also reduced saccharin SA in male Wistars and female msPs, suggesting the occurrence of some nonspecific effects at 60 mg/kg of the drug. Similarly, the highest dose of CORT113176 (60 mg/kg) decreased saccharin intake in male Wistars. Analysis of CORT levels revealed that females of both rat lines had higher blood levels of CORT compared to males. Alcohol consumption reduced CORT in females but not in males. Overall, these findings indicate that selective blockade of GR selectively reduces alcohol SA, and genetically selected msP rats are less sensitive to this pharmacological manipulation compared to heterogeneous Wistars. Moreover, results suggest sex differences in response to GR antagonism and the ability of alcohol to regulate GR transmission.

Neutrophils drive type I interferon production and autoantibodies in patients with Wiskott-Aldrich syndrome.

BACKGROUND: Wiskott-Aldrich syndrome (WAS) is a rare primary immunodeficiency caused by mutations in Wiskott-Aldrich syndrome protein (WASp), a key regulator of cytoskeletal dynamics in hematopoietic cells. A high proportion of patients experience autoimmunity caused by a breakdown in T- and B-cell tolerance. Moreover, excessive production of type I interferon (IFN-I) by plasmacytoid dendritic cells (pDCs) contributes to autoimmune signs; however, the factors that trigger excessive innate activation have not been defined. OBJECTIVE: Neutrophil extracellular traps (NETs) emerged as major initiating factors in patients with diseases such as systemic lupus erythematosus and rheumatoid arthritis. In this study we explored the possible involvement of aberrant neutrophil functions in patients with WAS. METHODS: We evaluated the expression of a set of granulocyte genes associated with NETs in a cohort of patients with WAS and the presence of NET inducers in sera. Using a mouse model of WAS, we analyzed NET release by WASp-null neutrophils and evaluated the composition and homeostasis of neutrophils in vivo. By using depletion experiments, we assessed the effect of neutrophils in promoting inflammation and reactivity against autoantigens. RESULTS: Transcripts of genes encoding neutrophil enzymes and antimicrobial peptides were increased in granulocytes of patients with WAS, and serum-soluble factors triggered NET release. WASp-null neutrophils showed increased spontaneous NETosis, induced IFN-I production by pDCs, and activated B cells through B-cell activating factor. Consistently, their depletion abolished constitutive pDC activation, normalized circulating IFN-I levels, and, importantly, abolished production of autoantibodies directed against double-stranded DNA, nucleosomes, and myeloperoxidase. CONCLUSIONS: These findings reveal that neutrophils are involved in the pathogenic loop that causes excessive activation of innate cells and autoreactive B cells, thus identifying novel mechanisms that contribute to the autoimmunity of WAS.

Control of murine Ly6C(high) monocyte traffic and immunosuppressive activities by atypical chemokine receptor D6.

The atypical chemokine receptor D6 is a decoy and scavenger receptor for most inflammatory CC chemokines and prevents the development of exacerbated inflammatory reactions. Here we report that mice lacking D6 expression in the nonhematopoietic compartment have a selective increase in the number of Ly6C(high) monocytes in the circulation and in secondary lymphoid tissues. Under inflammatory conditions, Ly6C(high) monocytes accumulate in increased number in secondary lymphoid organs of D6(-/-) mice in a CCR2-dependent manner. Ly6C(high) monocytes derived from D6(-/-) mice have enhanced immunosuppressive activity, inhibit the development of adaptive immune responses, and partially protect mice from the development of GVHD. Thus, control of CCR2 ligands by D6 regulates the traffic of Ly6C(high) monocytes and controls their immunosuppressive potential.

ALCAM-mediated cDC1 CD8 T cells interactions are suppressed in advanced lung tumors.

Conventional type 1 dendritic cells (cDC1) are critical regulators of anti-tumoral T-cell responses. The structure and abundance of intercellular contacts between cDC1 and CD8 T cells in cancer tissues is important to determine the outcome of the T-cell response. However, the molecular determinants controlling the stability of cDC1-CD8 interactions during cancer progression remain poorly investigated. Here, we generated a genetic model of non-small cell lung cancer crossed to a fluorescent cDC1 reporter (KP-XCR1venus) to allow the detection of cDC1-CD8T cell clusters in tumor tissues across tumor stages. We found that cDC1-CD8 clusters are abundant and productive at the early stages of tumor development but progressively diminish in advanced tumors. Transcriptional profiling and flow cytometry identified the adhesion molecule ALCAM/CD166 (Activated Leukocyte Cell Adhesion Molecule, ligand of CD6) as highly expressed by lung cDC1 and significantly downregulated in advanced tumors. Analysis of human datasets indicated that ALCAM is downregulated in non-small cell lung cancer and its expression correlates to better prognosis. Mechanistically, triggering ALCAM on lung cDC1 induces cytoskeletal remodeling and contact formation whereas its blockade prevents T-cell activation. Together, our results indicate that ALCAM is important to stabilize cDC1-CD8 interactions at early tumor stages, while its loss in advanced tumors contributes to immune evasion.

Cebranopadol, a novel long-acting opioid agonist with low abuse liability, to treat opioid use disorder: Preclinical evidence of efficacy.

Maintenance therapy with buprenorphine and methadone is the gold standard pharmacological treatment for opioid use disorder (OUD). Despite these compounds demonstrating substantial efficacy, a significant number of patients do not show optimal therapeutic responses. The abuse liability of these medications is also a concern. Here we used rats to explore the therapeutic potential of the new long-acting pan-opioid agonist Cebranopadol in OUD. We tested the effect of cebranopadol on heroin self-administration and yohimbine-induced reinstatement of heroin seeking. In addition, we evaluated the abuse liability potential of cebranopadol in comparison to that of heroin under fixed ratio 1 (FR1) and progressive ratio (PR) operant self-administration contingencies. Oral administration of cebranopadol (0, 25, 50 µg/kg) significantly attenuated drug self-administration independent of heroin dose (1, 7, 20, 60µg/inf). Cebranopadol also reduced the break point for heroin (20 µg/inf). Finally, pretreatment with cebranopadol significantly attenuated yohimbine-induced reinstatement of drug seeking. In abuse liability experiments under FR1 contingency, rats maintained responding for heroin (1, 7, 20, 60µg/inf) to a larger extent than cebranopadol (0.03, 0.1, 0.3, 1.0, 6.0µg/inf). Under PR contingency, heroin maintained responding at high levels at all except the lowest dose, while the break point (BP) for cebranopadol did not differ from that of saline. Together, these data indicate that cebranopadol is highly efficacious in attenuating opioid self-administration and stress-induced reinstatement, while having limited abuse liability properties. Overall, the data suggest clinical potential of this compound for OUD treatment.

Tim4 enables large peritoneal macrophages to cross-present tumor antigens at early stages of tumorigenesis.

Receptors controlling the cross-presentation of tumor antigens by macrophage subsets in cancer tissues are poorly explored. Here, we show that TIM4+ large peritoneal macrophages efficiently capture and cross-present tumor-associated antigens at early stages of peritoneal infiltration by ovarian cancer cells. The phosphatidylserine (PS) receptor TIM4 promotes maximal uptake of dead cells or PS-coated artificial targets and triggers inflammatory and metabolic gene programs in combination with cytoskeletal remodeling and upregulation of transcriptional signatures related to antigen processing. At the cellular level, TIM4-mediated engulfment induces nucleation of F-actin around nascent phagosomes, delaying the recruitment of vacuolar ATPase, acidification, and cargo degradation. In vivo, TIM4 deletion blunts induction of early anti-tumoral effector CD8 T cells and accelerates the progression of ovarian tumors. We conclude that TIM4-mediated uptake drives the formation of specialized phagosomes that prolong the integrity of ingested antigens and facilitate cross-presentation, contributing to immune surveillance of the peritoneum.

Dendritic cell-targeted therapy expands CD8 T cell responses to bona-fide neoantigens in lung tumors.

Cross-presentation by type 1 DCs (cDC1) is critical to induce and sustain antitumoral CD8 T cell responses to model antigens, in various tumor settings. However, the impact of cross-presenting cDC1 and the potential of DC-based therapies in tumors carrying varied levels of bona-fide neoantigens (neoAgs) remain unclear. Here we develop a hypermutated model of non-small cell lung cancer in female mice, encoding genuine MHC-I neoepitopes to study neoAgs-specific CD8 T cell responses in spontaneous settings and upon Flt3L + αCD40 (DC-therapy). We find that cDC1 are required to generate broad CD8 responses against a range of diverse neoAgs. DC-therapy promotes immunogenicity of weaker neoAgs and strongly inhibits the growth of high tumor-mutational burden (TMB) tumors. In contrast, low TMB tumors respond poorly to DC-therapy, generating mild CD8 T cell responses that are not sufficient to block progression. scRNA transcriptional analysis, immune profiling and functional assays unveil the changes induced by DC-therapy in lung tissues, which comprise accumulation of cDC1 with increased immunostimulatory properties and less exhausted effector CD8 T cells. We conclude that boosting cDC1 activity is critical to broaden the diversity of anti-tumoral CD8 T cell responses and to leverage neoAgs content for therapeutic advantage.

EMID2 is a novel biotherapeutic for aggressive cancers identified by in vivo screening.

BACKGROUND: New drugs to tackle the next pathway or mutation fueling cancer are constantly proposed, but 97% of them are doomed to fail in clinical trials, largely because they are identified by cellular or in silico screens that cannot predict their in vivo effect. METHODS: We screened an Adeno-Associated Vector secretome library (> 1000 clones) directly in vivo in a mouse model of cancer and validated the therapeutic effect of the first hit, EMID2, in both orthotopic and genetic models of lung and pancreatic cancer. RESULTS: EMID2 overexpression inhibited both tumor growth and metastatic dissemination, consistent with prolonged survival of patients with high levels of EMID2 expression in the most aggressive human cancers. Mechanistically, EMID2 inhibited TGFß maturation and activation of cancer-associated fibroblasts, resulting in more elastic ECM and reduced levels of YAP in the nuclei of cancer cells. CONCLUSION: This is the first in vivo screening, precisely designed to identify proteins able to interfere with cancer cell invasiveness. EMID2 was selected as the most potent protein, in line with the emerging relevance of the tumor extracellular matrix in controlling cancer cell invasiveness and dissemination, which kills most of cancer patients.

Cebranopadol, a novel long-acting opioid agonist with low abuse liability, to treat opioid use disorder: Preclinical evidence of efficacy.

The gold standard pharmacological treatment for opioid use disorder (OUD) consists of maintenance therapy with long-acting opioid agonists such as buprenorphine and methadone. Despite these compounds having demonstrated substantial efficacy, a significant number of patients do not show optimal therapeutic responses. Moreover, the abuse liability of these medications remains a major concern. Cebranopadol, is a new, long-acting pan-opioid agonist that also activates the nociception/orphanin FQ NOP receptor. Here we used rats to explore the therapeutic potential of this agent in OUD. First, in operant intravenous self-administration experiments we compared the potential abuse liability of cebranopadol with the prototypical opioid heroin. Under a fixed ratio 1 (FR1) contingency, rats maintained responding for heroin (1, 7, 20, 60 µg/inf) to a larger extent than cebranopadol (0.03, 0.1, 0.3, 1.0, 6.0 µg/inf). When the contingency was switched to a progressive ratio (PR) reinforcement schedule, heroin maintained responding at high levels at all except the lowest dose. Conversely, in the cebranopadol groups responding decreased drastically and the break point (BP) did not differ from saline controls. Next, we demonstrated that oral administration of cebranopadol (0, 25, 50 µg/kg) significantly attenuated drug self-administration independent of heroin dose (1, 7, 20, 60 µg/inf). Cebranopadol also reduced the break point for heroin (20 µg/inf). Furthermore, in a heroin self-administration training extinction/reinstatement paradigm, pretreatment with cebranopadol significantly attenuated yohimbine stress-induced reinstatement of drug seeking. Together, these data indicate that cebranopadol has limited abuse liability compared to heroin and is highly efficacious in attenuating opioid self-administration and stress-induced reinstatement, suggesting clinical potential of this compound for OUD treatment.

A mitochondrial quality control mechanism reverses the phagosome maturation arrest caused by Mycobacterium tuberculosis.

Phagosome maturation arrest (PMA) imposed by Mycobacterium tuberculosis ( Mtb ) is a classic tool that helps Mtb evade macrophage anti-bacterial responses. The exclusion of RAB7, a small GTPase, from Mtb -phagosomes underscores PMA. Here we report an unexpected mechanism that triggers crosstalk between the mitochondrial quality control (MQC) and the phagosome maturation pathways that reverses the PMA. CRISPR-mediated p62/SQSTM1 depletion ( p62 KD ) blocks mitophagy flux without impacting mitochondrial quality. In p62 KD cells, Mtb growth and survival are diminished, mainly through witnessing an increasingly oxidative environment and increased lysosomal targeting. The lysosomal targeting of Mtb is facilitated by enhanced TOM20 + mitochondria-derived vesicles (MDVs) biogenesis, a key MQC mechanism. In p62 KD cells, TOM20 + -MDVs biogenesis is MIRO1/MIRO2-dependent and delivered to lysosomes for degradation in a RAB7-dependent manner. Upon infection in p62 KD cells, TOM20 + -MDVs get extensively targeted to Mtb -phagosomes, inadvertently facilitating RAB7 recruitment, PMA reversal and lysosomal targeting of Mtb . Triggering MQC collapse in p62 KD cells further diminishes Mtb survival signifying cooperation between redox- and lysosome-mediated mechanisms. The MQC-anti-bacterial pathway crosstalk could be exploited for host-directed anti-tuberculosis therapies.

Ischemic wound revascularization by the stromal vascular fraction relies on host-donor hybrid vessels.

Nonhealing wounds place a significant burden on both quality of life of affected patients and health systems. Skin substitutes are applied to promote the closure of nonhealing wounds, although their efficacy is limited by inadequate vascularization. The stromal vascular fraction (SVF) from the adipose tissue is a promising therapy to overcome this limitation. Despite a few successful clinical trials, its incorporation in the clinical routine has been hampered by their inconsistent results. All these studies concluded by warranting pre-clinical work aimed at both characterizing the cell types composing the SVF and shedding light on their mechanism of action. Here, we established a model of nonhealing wound, in which we applied the SVF in combination with a clinical-grade skin substitute. We purified the SVF cells from transgenic animals to trace their fate after transplantation and observed that it gave rise to a mature vascular network composed of arteries, capillaries, veins, as well as lymphatics, structurally and functionally connected with the host circulation. Then we moved to a human-in-mouse model and confirmed that SVF-derived endothelial cells formed hybrid human-mouse vessels, that were stabilized by perivascular cells. Mechanistically, SVF-derived endothelial cells engrafted and expanded, directly contributing to the formation of new vessels, while a population of fibro-adipogenic progenitors stimulated the expansion of the host vasculature in a paracrine manner. These data have important clinical implications, as they provide a steppingstone toward the reproducible and effective adoption of the SVF as a standard care for nonhealing wounds.

CXCL5-mediated accumulation of mature neutrophils in lung cancer tissues impairs the differentiation program of anticancer CD8 T cells and limits the efficacy of checkpoint inhibitors.

Lung tumor-infiltrating neutrophils are known to support growth and dissemination of cancer cells and to suppress T cell responses. However, the precise impact of tissue neutrophils on programming and differentiation of anticancer CD8 T cells in vivo remains poorly understood. Here, we identified cancer cell-autonomous secretion of CXCL5 as sufficient to drive infiltration of mature, protumorigenic neutrophils in a mouse model of non-small cell lung cancer (NSCLC). Consistently, CXCL5 transcripts correlate with neutrophil density and poor prognosis in a large human lung adenocarcinoma compendium. CXCL5 genetic deletion, unlike antibody-mediated depletion, completely and selectively prevented neutrophils accumulation in lung tissues. Depletion of tumor-infiltrating neutrophils promoted expansion of tumor-specific CD8 T cells, differentiation into effector cells and acquisition of cytolytic functions. Transfer of effector CD8 T cells into neutrophil-rich tumors, inhibited IFN-ϒ production, indicating active suppression of effector functions. Importantly, blocking neutrophils infiltration in the lung, overcame resistance to checkpoint blockade. Hence, this study demonstrates that neutrophils curb acquisition of cytolytic functions in lung tumor tissues and suggests targeting of CXCL5 as a strategy to restore anti-tumoral T cell functions.