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BACKGROUND INFORMATION:Cell motility entails the reorganization of the cytoskeleton and membrane trafficking for effective protrusion. The GIT-PIX protein complexes are involved in the regulation of cell motility and adhesion and in the endocytic traffic of members of the family of G-protein-coupled receptors. We have investigated the function of the endogenous GIT complexes in the regulation of cell motility stimulated by fMLP (formyl-Met-Leu-Phe) peptide, in a rat basophilic leukaemia RBL-2H3 cell line stably expressing an HA (haemagglutinin)-tagged receptor for the fMLP peptide.RESULTS:Our analysis shows that RBL cells stably transfected with the chemoattractant receptor expressed both GIT1-PIX and GIT2-PIX endogenous complexes. We have used silencing of the different members of the complex by small interfering RNAs to study the effects on a number of events linked to agonist-induced cell migration. We found that cell adhesion was not affected by depletion of any of the proteins of the GIT complex, whereas agonist-enhanced cell spreading was inhibited. Analysis of agonist-stimulated haptotactic cell migration indicated a specific positive effect of GIT1 depletion on trans-well migration. The internalization of the formyl-peptide receptor was also inhibited by depletion of GIT1 and GIT2. The effects of the GIT complexes on trafficking of the receptors was confirmed by an antibody-enhanced agonist-induced internalization assay, showing that depletion of PIX, GIT1 or GIT2 protein caused decreased perinuclear accumulation of internalized receptors.CONCLUSIONS:Our results show that endogenous GIT complexes are involved in the regulation of chemoattractant-induced cell motility and receptor trafficking, and support previous findings indicating an important function of the GIT complexes in the regulation of different G-protein-coupled receptors. Our results also indicate that endogenous GIT1 and GIT2 regulate distinct subsets of agonist-induced responses and suggest a possible functional link between the control of receptor trafficking and the regulation of cell motility by GIT proteins.

Inhibitor of NF-kappaB kinases beta (IKKbeta) and alpha (IKKalpha) activate distinct NF-kappaB signaling modules. The IKKbeta/canonical NF-kappaB pathway rapidly responds to stress-like conditions, whereas the IKKalpha/noncanonical pathway controls adaptive immunity. Moreover, IKKalpha can attenuate IKKbeta-initiated inflammatory responses. High mobility group box 1 (HMGB1), a chromatin protein, is an extracellular signal of tissue damage-attracting cells in inflammation, tissue regeneration, and scar formation. We show that IKKalpha and IKKbeta are each critically important for HMGB1-elicited chemotaxis of fibroblasts, macrophages, and neutrophils in vitro and neutrophils in vivo. By time-lapse microscopy we dissected different parameters of the HMGB1 migration response and found that IKKalpha and IKKbeta are each essential to polarize cells toward HMGB1 and that each kinase also differentially affects cellular velocity in a time-dependent manner. In addition, HMGB1 modestly induces noncanonical IKKalpha-dependent p52 nuclear translocation and p52/RelB target gene expression. Akin to IKKalpha and IKKbeta, p52 and RelB are also required for HMGB1 chemotaxis, and p52 is essential for cellular orientation toward an HMGB1 gradient. RAGE, a ubiquitously expressed HMGB1 receptor, is required for HMGB1 chemotaxis. Moreover, IKKbeta, but not IKKalpha, is required for HMGB1 to induce RAGE mRNA, suggesting that RAGE is at least one IKKbeta target involved in HMGB1 migration responses, and in accord with these results enforced RAGE expression rescues the HMGB1 migration defect of IKKbeta, but not IKKalpha, null cells. Thus, proinflammatory HMGB1 chemotactic responses mechanistically require the differential collaboration of both IKK-dependent NF-kappaB signaling pathways.

Unlike most somatic cells, leukocytes are constitutively non-adherent. However, adhesive interactions are not only a required step in essentially all effector functions performed by leukocytes, but they also relay increasingly well-defined intracellular signals that affect the leukocyte as well as the surrounding tissues. Dissecting such signals in leukocytes has provided a wealth of information that contributes to our understanding of how adhesion controls higher-order biological responses, ranging from cell migration to proliferation, differentiation and survival.

Cell migration entails the dynamic redistribution of adhesion receptors from the cell rear toward the cell front, where they form new protrusions and adhesions. This process may involve regulated endo-exocytosis of integrins. Here we show that in primary neutrophils unengaged alphaL/beta2 integrin (LFA-1) is internalized and rapidly recycled upon chemoattractant stimulation via a clathrin-independent, cholesterol-sensitive pathway involving dynamic partitioning into detergent-resistant membranes (DRM). Persistent DRM association is required for recycling of the internalized receptor because 1) >90% of endocytosed LFA-1 is associated with DRM, and a large fraction of the internalized receptor colocalizes intracellularly with markers of DRM and the recycling endocytic compartment; 2) a recycling-defective mutant (alphaL/beta2Y735A) dissociates rapidly from DRM upon being endocytosed and is subsequently diverted into a late endosomal pathway; and 3) a dominant negative Rab11 mutant (Rab11S25N) induces intracellular accumulation of endocytosed alphaL/beta2 and prevents its enrichment in chemoattractant-induced lamellipodia. Notably, chemokine-induced migration of neutrophils over immobilized ICAM-1 is abrogated by cholesterol-sequestering agents. We propose that DRM-associated endocytosis allows efficient retrieval of integrins, as they detach from their ligands, followed by polarized recycling to areas of the plasma membrane, such as lamellipodia, where they establish new adhesive interactions and promote outside-in signaling events.

Background Multi-element interventions for first-episode psychosis (FEP) are promising, but have mostly been conducted in non-epidemiologically representative samples, thereby raising the risk of underestimating the complexities involved in treating FEP in ‘real-world’ services. Methods/Design The Psychosis early Intervention and Assessment of Needs and Outcome (PIANO) trial is part of a larger research program (Genetics, Endophenotypes and Treatment: Understanding early Psychosis - GET UP) which aims to compare, at 9 months, the effectiveness of a multi-component psychosocial intervention versus treatment as usual (TAU) in a large epidemiologically based cohort of patients with FEP and their family members recruited from all public community mental health centers (CMHCs) located in two entire regions of Italy (Veneto and Emilia Romagna), and in the cities of Florence, Milan and Bolzano. The GET UP PIANO trial has a pragmatic cluster randomized controlled design. The randomized units (clusters) are the CMHCs, and the units of observation are the centers’ patients and their family members. Patients in the experimental group will receive TAU plus: 1) cognitive behavioral therapy sessions, 2) psycho-educational sessions for family members, and 3) case management. Patient enrolment will take place over a 1-year period. Several psychopathological, psychological, functioning, and service use variables will be assessed at baseline and follow-up. The primary outcomes are: 1) change from baseline to follow-up in positive and negative symptoms’ severity and subjective appraisal; 2) relapse occurrences between baseline and follow-up, that is, episodes resulting in admission and/or any case-note records of re-emergence of positive psychotic symptoms. The expected number of recruited patients is about 400, and that of relatives about 300. Owing to the implementation of the intervention at the CMHC level, the blinding of patients, clinicians, and raters is not possible, but every effort will be made to preserve the independency of the raters. We expect that this study will generate evidence on the best treatments for FEP, and will identify barriers that may hinder its feasibility in ‘real-world’ clinical settings, patient/family conditions that may render this intervention ineffective or inappropriate, and clinical, psychological, environmental, and service organization predictors of treatment effectiveness, compliance, and service satisfaction. Trial registration ClinicalTrials.gov Identifier NCT01436331

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ObjectiveTrained immunity (TI) is a de facto memory program of innate immune cells, characterized by immunometabolic and epigenetic changes sustaining enhanced production of cytokines. TI evolved as a protective mechanism against infections; however, inappropriate activation can cause detrimental inflammation and might be implicated in the pathogenesis of chronic inflammatory diseases. In this study, we investigated the role of TI in the pathogenesis of giant cell arteritis (GCA), a large-vessel vasculitis characterized by aberrant macrophage activation and excess cytokine production.MethodsMonocytes from GCA patients and from age- and sex-matched healthy donors were subjected to polyfunctional studies, including cytokine production assays at baseline and following stimulation, intracellular metabolomics, chromatin immunoprecipitation-qPCR, and combined ATAC/RNA sequencing. Immunometabolic activation (i.e. glycolysis) was assessed in inflamed vessels of GCA patients with FDG-PET and immunohistochemistry (IHC), and the role of this pathway in sustaining cytokine production was confirmed with selective pharmacologic inhibition in GCA monocytes.ResultsGCA monocytes exhibited hallmark molecular features of TI. Specifically, these included enhanced IL-6 production upon stimulation, typical immunometabolic changes (e.g. increased glycolysis and glutaminolysis) and epigenetic changes promoting enhanced transcription of genes governing pro-inflammatory activation. Immunometabolic changes of TI (i.e. glycolysis) were a feature of myelomonocytic cells in GCA lesions and were required for enhanced cytokine production.ConclusionsMyelomonocytic cells in GCA activate TI programs sustaining enhanced inflammatory activation with excess cytokine production.

Summary Trained immunity (TI) is a de facto innate immune memory program induced in monocytes/macrophages by exposure to pathogens or vaccines, which evolved as protection against infections. TI is characterized by immunometabolic changes and histone post-translational modifications, which enhance production of pro-inflammatory cytokines. As aberrant activation of TI is implicated in inflammatory diseases, tight regulation is critical; however, the mechanisms responsible for this modulation remain elusive. Interleukin-37 (IL-37) is an anti-inflammatory cytokine that curbs inflammation and modulates metabolic pathways. In this study, we show that administration of recombinant IL-37 abrogates the protective effects of TI in vivo, as revealed by reduced host pro-inflammatory responses and survival to disseminated candidiasis. Mechanistically, IL-37 reverses the immunometabolic changes and histone post-translational modifications characteristic of TI in monocytes, thus suppressing cytokine production in response to infection. IL-37 thereby emerges as an inhibitor of TI and as a potential therapeutic target in immune-mediated pathologies.

Alzheimer’s disease (AD) is the most frequent type of dementia in older people. The complex nature of AD calls for the development of multitarget agents addressing key pathogenic processes. Donepezil, an acetylcholinesterase inhibitor, is a first-line acetylcholinesterase inhibitor used for the treatment of AD. Although several studies have demonstrated the symptomatic efficacy of donepezil treatment in AD patients, the possible effects of donepezil on the AD process are not yet known. In this study, a novel feruloyl–donepezil hybrid compound (PQM130) was synthesized and evaluated as a multitarget drug candidate against the neurotoxicity induced by Aβ1-42 oligomer (AβO) injection in mice. Interestingly, PQM130 had already shown anti-inflammatory activity in different in vivo models and neuroprotective activity in human neuronal cells. The intracerebroventricular (i.c.v.) injection of AβO in mice caused the increase of memory impairment, oxidative stress, neurodegeneration, and neuroinflammation. Instead, PQM130 (0.5–1 mg/kg) treatment after the i.c.v. AβO injection reduced oxidative damage and neuroinflammation and induced cell survival and protein synthesis through the modulation of glycogen synthase kinase 3β (GSK3β) and extracellular signal–regulated kinases (ERK1/2). Moreover, PQM130 increased brain plasticity and protected mice against the decline in spatial cognition. Even more interesting is that PQM130 modulated different pathways compared to donepezil, and it is much more effective in counteracting AβO damage. Therefore, our findings highlighted that PQM130 is a potent multi-functional agent against AD and could act as a promising neuroprotective compound for anti-AD drug development.

Major depressive disorder is one of the most prevalent and life-threatening forms of mental illnesses and a major cause of morbidity worldwide. Currently available antidepressants are effective for most patients, although around 30% are considered treatment resistant (TRD), a condition that is associated with a significant impairment of cognitive function and poor quality of life. In this respect, the identification of the molecular mechanisms contributing to TRD represents an essential step for the design of novel and more efficacious drugs able to modify the clinical course of this disorder and increase remission rates in clinical practice. New insights into the neurobiology of TRD have shed light on the role of a number of different mechanisms, including the glutamatergic system, immune/inflammatory systems, neurotrophin function, and epigenetics. Advances in drug discovery processes in TRD have also influenced the classification of antidepressant drugs and novel classifications are available, such as the neuroscience-based nomenclature that can incorporate such advances in drug development for TRD. This review aims to provide an up-to-date description of key mechanisms in TRD and describe current therapeutic strategies for TRD before examining novel approaches that may ultimately address important neurobiological mechanisms not targeted by currently available antidepressants. All in all, we suggest that drug targeting different neurobiological systems should be able to restore normal function but must also promote resilience to reduce the long-term vulnerability to recurrent depressive episodes.

The neurovascular unit (NVU) is the most important biological barrier between vascular districts and central nervous system (CNS) parenchyma, which maintains brain homeostasis, protects the CNS from pathogens penetration, and mediates neuroimmune communication. T lymphocytes migration across the blood-brain barrier is heavily affected in different brain diseases, representing a major target for novel drug development. In vitro models of NVU could represent a primary tool to investigate the molecular events occurring at this interface. To move toward the establishment of personalized therapies, a patient-related NVU-model is set, incorporating human primary astrocytes integrated into a microfluidic platform. The model is morphologically and functionally characterized, proving to be an advantageous tool to investigate human T lymphocytes transmigration and thus the efficacy of potential novel drugs affecting this process.

Recruitment of leukocytes from blood to tissues is a multi-step process playing a major role in the activation of inflammatory responses. Tethering and rolling of leukocytes along the vessel wall, followed by arrest and transmigration through the endothelium result from chemoattractant-dependent signals, inducing adhesive and migratory events. Shear forces exerted by the blood flow on leukocytes induce rolling via selectin-mediated interactions with endothelial cells and increase the probability of leukocytes to engage their chemokine receptors, facilitating integrin activation and consequent arrest. Flow-derived shear forces generate mechanical stimuli concurring with biochemical signals in the modulation of leukocyte-endothelial cell interactions. In the last few years, a host of in vitro studies have clarified the biochemical adhesion cascade and the role of shear stress in leukocyte extravasation. The limitation of the static environment in Boyden devices has been overcome both by the use of parallel-plate flow chambers and by custom models mimicking the in vivo conditions, along with widespread microfluidic approaches to in vitro modeling. These devices create an in vitro biomimetic environment where the multi-step transmigration process can be imaged and quantified under mechanical and biochemical controlled conditions, including fluid dynamic settings, channel design, materials and surface coatings. This paper reviews the technological solutions recently proposed to model, observe and quantify leukocyte adhesion behavior under shear flow, with a final survey of high-throughput solutions featuring multiple parallel assays as well as thorough and time-saving statistical interpretation of the experimental results. (c) 2012 Elsevier Ltd. All rights reserved.

Objectives. The association between depression and circadian rhythm disturbances is well established and successful treatment of depressed patients is accompanied by restoration of circadian rhythms. The new antidepressant agomelatine is an agonist of melatonergic MT(1)/MT(2) receptors as well as an antagonist of serotonergic 5-HT(2C) receptors. Animal studies showed that agomelatine resynchronizes disturbed circadian rhythms and reduces depression-like behaviour. Methods. This review analyzes results from different experimental studies. Results. Recent data on the effects of agomelatine on cellular processes involved in antidepressant mechanisms have shown that the drug is able to increase the expression of brain-derived neurotrophic factor in prefrontal cortex and hippocampus, as well as the expression of activity-regulated cytoskeleton associated protein (Arc) in the prefrontal cortex. In line with this, prolonged treatment with agomelatine increases neurogenesis within the hippocampus, particularly via enhancement of neuronal cell survival. Agomelatine attenuates stress-induced glutamate release in the prefrontal/frontal cortex. Treatment with 5-HT(2C) antagonists or melatonin alone failed to reproduce these effects. Conclusions. The unique mode of action of agomelatine may improve the management of major depression by counteracting the pathogenesis of depression at cellular level, thereby relieving the symptoms of depression. These effects are suggested to be due to a synergistic action on MT(1)/MT(2) and 5-HT(2C) receptors.

Objectives. Alterations of BDNF signalling in major depression (MD) are supported by studies demonstrating decreased levels of the neurotrophin serum and plasma content in MD patients. We conducted a replication study and we performed two meta-analyses on studies analysing serum and plasma BDNF levels in MD patients. Methods. The samples were composed by 489 patients/483 controls for the meta-analysis on serum and by 161 patients/211 controls for that on plasma levels. We performed also subgroup analyses to examine whether the decrease in BDNF levels in MD was influenced by gender. Results. In the replication study we found decreased serum BDNF levels in MD patients (P

Leukocyte extravasation involves interdependent signaling pathways underlying the complex dynamics of firm adhesion, crawling, and diapedesis. While signal transduction by agonist-bound chemokine receptors plays a central role in the above responses, it is unclear how it contributes to the sustained and concurrent nature of such responses, given the rapid kinetics of chemokine-induced trimeric G protein coupling and homologous desensitization. Our findings unveil a novel role of beta-arrestins in regulating the activation of signaling pathways underlying discrete integrin-mediated steps in CXCR2-driven leukocyte extravasation. By combining in vivo approaches in beta-arrestin knockout mice with in vitro studies in engineered cellular models, we show that membrane-recruited beta-arrestin 2 is required for the onset and maintenance of shear stress-resistant leukocyte adhesion mediated by both beta(1) and beta(2) integrins. While both beta-arrestin isoforms are required for rapid keratinocyte-derived chemokine (KC)-induced arrest onto limiting amounts of vascular cell adhesion molecule-1 (VCAM-1), adhesion strengthening under shear is selectively dependent on beta-arrestin 2. The latter synergizes with phospholipase C in promoting activation of Rap1A and B, both of which co-operatively control subsecond adhesion as well as postarrest adhesion stabilization. Thus, receptor-induced Galpha(i) and beta-arrestins act sequentially and in spatially distinct compartments to promote optimal KC-induced integrin-dependent adhesion during leukocyte extravasation.

During the last few years several studies have highlighted the possibility that major depression can be characterized by a general reduction in brain plasticity and an increased vulnerability under challenging situations. Such dysfunction may be the consequence of reduced expression and function of proteins important for neuroplasticity such as brain-derived neurotrophic factor (BDNF). On this basis, by using a sensitive non-radioactive in-situ hybridization, we evaluated the effects of a chronic treatment with fluoxetine on BDNF expression within rat dopaminergic regions. In fact, besides the well-established role of the hippocampus, increasing evidence indicates that other brain regions may be involved in the pathophysiology of depression and consequently be relevant for the therapeutic action of antidepressant drugs. Our results indicate that 3 wk of fluoxetine administration up-regulates BDNF mRNA levels selectively within structures belonging to the meso-cortico-limbic pathway. The expression of the neurotrophin is significantly increased in the ventral tegmental area, prefrontal cortex, and shell region of the nucleus accumbens, whereas no changes were detected in the substantia nigra and striatum. Moreover, in agreement with previous studies, fluoxetine increased BDNF mRNA levels in the hippocampus, an effect that was limited to the cell bodies without any change in its dendritic targeting. These data show that chronic treatment with fluoxetine increases BDNF gene expression not only in limbic areas but also in dopaminergic regions, suggesting that such an effect may contribute to improve the function of the dopaminergic system in depressed subjects.