Endocannabinoids modulate neuroglial phenotype and proteotoxic stress

ABSTRACT Neuronal survival in neurodegenerative diseases and brain damage is closely related to the cell populations of the environment and in particular to glial cells. Astrocytes, microglia and oligodendrocytes oversee brain homeostasis providing the intrinsic brain defence system. Damage to brain cells triggers a condition generally referred to as reactive gliosis, which includes astrogliosis and activation of microglia. Neuroglia is also thoroughly involved in pathogenesis of many chronic neurological disorders and in neurodegeneration. Endocannabinoids modulating the behaviour of microglia and astrocytes might act as possible targets for therapeutic intervention. Recent studies have indicated that endocannabinoid levels and metabolic enzymes change during the progression of Alzheimer's disease (AD) and that the inhibition of fatty acid amide hydrolase (FAAH), the main catabolic enzyme of anandamide (AEA), has beneficial effects in mice with AD. The aim of this study was to determine whether URB597, a FAAH inhibitor, targets microglia polarization by altering the cytoskeleton reorganization induced by amyloid-β peptide (Aβ) in BV-2 microglial cells. Evaluation of actin cytoskeleton showed that Aβ treatment increased the surface area of BV-2 cells, which acquired a flat and polygonal morphology. Although URB597 did not affect cell morphology only, it partially rescued the control phenotype in BV-2 cells incubated with the combined treatment. Rho family proteins have a critical role in the plasticity of the actin cytoskeleton, influencing morphological changes, migration and phagocytic activity of cells. We observed an increase of Rho protein activation in Aβ samples and a decrease in samples treated with URB597 alone or in combination with Aβ compared to controls, while an increase of Cdc42 protein activation was observed in all samples with respect to control. Aβ induced the migration of BV-2 cells up to 2 h after stimulation. We also found that by reducing Rho protein activity, URB597 was able to reduce the migration rate. URB597 also increased the number of BV-2 cells performing phagocytosis. Taken together, these data suggest that an increase of anandamide (AEA), due to FAAH inhibition, may induce cytoskeleton reorganization, regulating phagocytosis and cell migration processes, and promote microglial polarization towards an anti-inflammatory phenotype. As most research worldwide has focused on neurons, there is a dearth of protocols to generate glial cells and to produce co-culture systems for biomedical research. The aim of this project has also been the generation of co-culture with neurons, astrocytes and microglia cells and the subsequent characterization of the resulting model, evaluating interspecies differences through the generation of co-cultures with murine microglia. We focused our interest on the repair functions during brain injury and on the interactions between microglia and astrocytes. The protective effect of astrocytes and microglia against neuronal cells in the presence of inflammatory and pro-apoptotic processes was investigated. Human astrocytes and human microglia cells were activated with TNF-, IL-1 and IFN- to evaluate the inflammatory response. The results showed an increase of inflammatory cytokines gene expression such as IL-6 and IL-8 in both cell lines examined. The astrocytes activation by TNF-, or by conditioned medium (CM) of activated microglia cells was confirmed by NF-kB nuclearization. Therefore, the arise of inflammatory process in astrocyte cells is driven not only by TNF- induction, but also by a synergic effect due to microglia activation. Neuroinflammation, oxidative stress, and progressive degeneration of specific brain regions is also driven by proteasomal impairment, promoting protein accumulations. Since LUHMES neurons are quite susceptible cells to proteotoxic stress and amino acid starvation, we investigated whether murine microglia and human astrocytes exerted a protective effect also when the cell lines were treated with URB597. The obtained data demonstrated that the astrocytes through the glutathione (GSH) release, were able to attenuate neuronal proteotoxic stress in LUHMES cells. URB597 contributed to GSH anti-oxidant effects modulating GSH metabolism. The overall data demonstrated that neuroglial cells play a pivotal role on neuronal protection from noxious stimuli

Immunophenotyping of peripheral blood cells allows to discriminate MIS-C and Kawasaki disease

Background: The pathogenesis of the novel described multisystem inflammatory syndrome in children (MIS-C) and Kawasaki disease (KD) is still debated as it is not clear if they are the same or different nosological entities. However, for both the diseases a rapid and unequivocal diagnosis is mandatory to start the therapy before the onset of severe complications. In this study, we aimed to evaluate the white cell populations in MIS-C and KD as potential markers to discriminate between the two diseases. Methods: We studied white cell populations by flow cytometry in 46 MIS-C and 28 KD patients in comparison to 70 age-matched healthy children. Results: MIS-C patients had a significant lymphopenia that involved both B and T populations while KD patients showed a significant neutrophilia and thrombocythemia. Granulocyte/lymphocyte ratio helped to diagnose both MIS-C and KD with a high diagnostic sensitivity, while a multivariate analysis of granulocyte and T lymphocyte number contributed to discriminate between the two diseases. Conclusions: The relevant lymphopenia observed in MIS-C patients suggests that the disease would be a post-infectious sequel of COVID-19 immunologically amplified by a massive cytokine release, while the significant neutrophilia and thrombocythemia observed in KD confirmed that the disorder has the genesis of a systemic vasculitis. The analysis of a panel of circulating cells may help to early diagnose and to discriminate between the two diseases. Supplementary information: The online version contains supplementary material available at 10.1186/s41231-022-00128-2

ERp57 chaperon protein protects neuronal cells from Aβ-induced toxicity

Alzheimer's disease (AD) is a neurodegenerative disorder whose main pathological hallmark is the accumulation of Amyloid-β peptide (Aβ) in the form of senile plaques. Aβ can cause neurodegeneration and disrupt cognitive functions by several mechanisms, including oxidative stress. ERp57 is a protein disulfide isomerase involved in the cellular stress response and known to be present in the cerebrospinal fluid of normal individuals as a complex with Aβ peptides, suggesting that it may be a carrier protein which prevents aggregation of Aβ. Although several studies show ERp57 involvement in neurodegenerative diseases, no clear mechanism of action has been identified thus far. In this work, we gain insights into the interaction of Aβ with ERp57, with a special focus on the contribution of ERp57 to the defense system of the cell. Here, we show that recombinant ERp57 directly interacts with the Aβ25-35 fragment in vitro with high affinity via two in silico-predicted main sites of interaction. Furthermore, we used human neuroblastoma cells to show that short-term Aβ25-35 treatment induces ERp57 decrease in intracellular protein levels, different intracellular localization, and ERp57 secretion in the cultured medium. Finally, we demonstrate that recombinant ERp57 counteracts the toxic effects of Aβ25-35 and restores cellular viability, by preventing Aβ25-35 aggregation. Overall, the present study shows that extracellular ERp57 can exert a protective effect from Aβ toxicity and highlights it as a possible therapeutic tool in the treatment of AD

Modulation of Methacrylated Hyaluronic Acid Hydrogels Enables Their Use as 3D Cultured Model

: Bioengineered hydrogels represent physiologically relevant platforms for cell behaviour studies in the tissue engineering and regenerative medicine fields, as well as in in vitro disease models. Hyaluronic acid (HA) is an ideal platform since it is a natural biocompatible polymer that is widely used to study cellular crosstalk, cell adhesion and cell proliferation, and is one of the major components of the extracellular matrix (ECM). We synthesised chemically modified HA with photo-crosslinkable methacrylated groups (HA-MA) in aqueous solutions and in strictly monitored pH and temperature conditions to obtain hydrogels with controlled bulk properties. The physical and chemical properties of the different HA-MA hydrogels were investigated via rheological studies, mechanical testing and scanning electron microscopy (SEM) imaging, which allowed us to determine the optimal biomechanical properties and develop a biocompatible scaffold. The morphological evolution processes and proliferation rates of glioblastoma cells (U251-MG) cultured on HA-MA surfaces were evaluated by comparing 2D structures with 3D structures, showing that the change in dimensionality impacted cell functions and interactions. The cell viability assays and evaluation of mitochondrial metabolism showed that the hydrogels did not interfere with cell survival. In addition, morphological studies provided evidence of cell-matrix interactions that promoted cell budding from the spheroids and the invasiveness in the surrounding environment

MIS-C: A COVID-19-associated condition between hypoimmunity and hyperimmunity

: Multisystem inflammatory syndrome in children (MIS-C) is a rare, severe complication of COVID-19. A better knowledge of immunological, cellular, and genetic characteristics of MIS-C could help better understand the pathogenesis of the disease and contribute to identifying specific diagnostic biomarkers and develop targeted therapies. We studied 37 MIS-C children at hospital admission and 24 healthy controls analyzing serum cytokines (IFN-α, IFN-β, IFN-γ, IL-6, IL-10, IL-17A, IL-12p70 and TNF), lymphocyte populations by flow cytometry and 386 genes related to autoimmune diseases, autoinflammation and primary immunodeficiencies by NGS. MIS-C patients showed a significant increase of serum IFNγ (despite a significant reduction of activated Th1) and ILs, even if with a great heterogeneity among patients, revealing different pathways involved in MIS-C pathogenesis and suggesting that serum cytokines at admission may help to select the inflammatory pathways to target in each patient. Flow cytometry demonstrated a relevant reduction of T populations while the percentage of B cell was increased in agreement with an autoimmune pathogenesis of MIS-C. Genetic analysis identified variants in 34 genes and 83.3% of patients had at least one gene variant. Among these, 9 were mutated in more patients. Most genes are related to autoimmune diseases like ATM, NCF1, MCM4, FCN3, and DOCK8 or to autoinflammatory diseases associated to the release of IFNγ like PRF1, NOD2, and MEF. Thus, an incomplete clearance of the Sars-CoV2 during the acute phase may induce tissue damage and self-antigen exposure and genetic variants can predispose to hyper-reactive immune dysregulation events of MIS-C-syndrome. Type II IFN activation and cytokine responses (mainly IL-6 and IL-10) may cause a cytokine storm in some patients with a more severe acute phase of the disease, lymphopenia and multisystemic organ involvement. The timely identification of such patients with an immunocytometric panel might be critical for targeted therapeutic management

Short-, mid- and long-term efficacy of dupilumab in moderate to severe atopic dermatitis: a real life multicenter Italian study on 2576 patients

Background: The efficacy and safety of dupilumab in atopic dermatitis (AD) have been defined in clinical trials but limited real-world evidence on long term treatment outcomes are currently available to inform clinical decisions. Objectives: to describe long-term effectiveness and safety of dupilumab up to 48 months in patients with moderate-to-severe AD. Methods: a multicenter, retrospective, dynamic cohort study was conducted to assess long term effectiveness and safety of dupilumab in patients with moderate to severe AD in a real-world setting. Predictors of minimal disease activity (MDA) optimal treatment target criteria (defined as the simultaneous achievement of EASI90, itch NRS score ≤1, sleep NRS score ≤1 and DLQI ≤1) were investigated. Results: 2576 patients were enrolled from June 2018 to July 2022. MDA optimal treatment target criteria were achieved by 506 (21.91%), 769 (40.63%), 628 (50.36%), 330 (55.37%) and 58 (54.72%) of those that reached 4, 12, 24, 36 and 48 months of follow-up, respectively. Logistic regression revealed a negative effect on MDA achievement for conjunctivitis and food allergy at all timepoints. Adverse events (AE) were mild and were observed in 373 (15.78%), 166 (7.02%), 83 (6.43%), 27 (4.50%) and 5 (4.55%) of those that reached 4, 12, 24, 36 and 48 months of follow-up. Conjunctivitis was the most frequently reported AE during the available follow-up. AE led to treatment discontinuation in <1% of patients during the evaluated time periods. Conclusion: High long-term effectiveness and safety of dupilumab were confirmed in this dynamic cohort of patients with moderate to severe AD, regardless of clinical phenotype and course at baseline. Further research will be needed to investigate the effect of Th2 comorbidities and disease duration on the response to dupilumab and other newer therapeutics for AD

HYDRHA:Hydrogels of hyaluronic acid. New biomedical approaches in cancer, neurodegenerative diseases, and tissue engineering

In the last decade, hyaluronic acid (HA) has attracted an ever-growing interest in the biomedical engineering field as a biocompatible, biodegradable, and chemically versatile molecule. In fact, HA is a major component of the extracellular matrix (ECM) and is essential for the maintenance of cellular homeostasis and crosstalk. Innovative experimental strategies in vitro and in vivo using three-dimensional (3D) HA systems have been increasingly reported in studies of diseases, replacement of tissue and organ damage, repairing wounds, and encapsulating stem cells for tissue regeneration. The present work aims to give an overview and comparison of recent work carried out on HA systems showing advantages, limitations, and their complementarity, for a comprehensive characterization of their use. A special attention is paid to the use of HA in three important areas: cancer, diseases of the central nervous system (CNS), and tissue regeneration, discussing the most innovative experimental strategies. Finally, perspectives within and beyond these research fields are discussed

COVID-19 infection and BNT162b2 vaccine triggering sarcoid-like lesions in the same patient Response to: Sarcoid-like reaction in a patient recovering from coronavirus disease 19 pneumonia

Dear Editor, Behbahani et al. recently reported on your journal a rare case of sarcoid granulomas induced by cross- reactivity to COVID-19 infection.1 Since then, different types of COVID-19 vaccines have been produced. Among these, BNT162b2 (Comirnaty®, Pfizer/BioNTech, New York, NY, USA) is an mRNA vaccine encoding the RBD of SARS-CoV-2 spike protein.2 We report a case of cutaneous sarcoid-like granulomatous reaction in a COVID-19 patient, reactivated by the administration of BNT162b2 vaccine

STAT3/ERP57/TPX2 axis and process of “androgen escape” in prostate cancer

The mechanisms of Prostate Cancer (PCa) progression through hormone-dependent to hormone refractory form is still unclear. Many data indicate that JAK/STAT signaling contributes to tumor resistance and STAT3 hyperactivation is observed in a variety of human cancers. Moreover, several authors suggested that ERp57 (GRP58/PDIA3), a disulfide isomerases protein, is associated with modulation of STAT3 activity. We investigate the role of STAT3-ERp57-TPX2 axis in the ormone-responsive and androgen-refractory tumor using human PCa cell lines, LNCaP (androgen-sensitive) and PC3 (androgen-refractory), untreated and stimulated with IL-6 and EGF. Immunoblotting and CoIP analysis were performed to confirm STAT3 activation and ERp57-STAT3 interaction. To investigate the physiological relevance of STAT3-ERp57-TPX2 axis, we inhibited STAT3 or ERp57 activity and the expression levels of TPX2 was monitored by qRT-PCR. The results showed that increased STAT3-ERp57 complex association determines an TPX2 overexpression. In conclusion, this study showed that STAT3-ERp57-TPX2 axis is correlated with tumor progression and it suggests a functional role of STAT3/ERp57 complex in the Androgen Escape