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We demonstrated that γδ T cells of patients given HLA-haploidentical HSCT after removal of αβ+T cells and CD19+B cells are endowed with the capacity of killing leukemia cells afterex vivotreatment with zoledronic acid (ZOL). Thus, we tested the hypothesis that infusion of ZOL in patients receiving this type of graft may enhance γδ T-cell cytotoxic activity against leukemia cells. ZOL was infused every 28 d in 43 patients; most were treated at least twice. γδ T cells before and after ZOL treatments were studied in 33 of these 43 patients, till at least 7 mo after HSCT by high-resolution mass spectrometry, flow-cytometry, and degranulation assay. An induction of Vδ2-cell differentiation, paralleled by increased cytotoxicity of both Vδ1 and Vδ2 cells against primary leukemia blasts was associated with ZOL treatment. Cytotoxic activity was further increased in Vδ2 cells, but not in Vδ1 lymphocytes in those patients given more than one treatment. Proteomic analysis of γδ T cells purified from patients showed upregulation of proteins involved in activation processes and immune response, paralleled by downregulation of proteins involved in proliferation. Moreover, a proteomic signature was identified for each ZOL treatment. Patients given three or more ZOL infusions had a better probability of survival in comparison to those given one or two treatments (86% vs. 54%, respectively,p= 0.008). Our data indicate that ZOL infusion in pediatric recipients of αβ T- and B-cell-depleted HLA-haploidentical HSCT promotes γδ T-cell differentiation and cytotoxicity and may influence the outcome of patients. [ABSTRACT FROM AUTHOR]

Objectives: To examine the prevalence of temporomandibular disorders (TMD) in patients with juvenile fibromyalgia syndrome (JFS) and identify TMD characteristics specifically associated to JFS., Methods: Signs and symptoms of TMD were assessed using a novel clinical tool specifically devised for children that consists of: 1. a self-report multiple-choice questionnaire; 2. a protocol for the clinical examination of the orofacial region. Multivariate logistic regression model was used to identify TMD features associated with JFS., Results: Thirty JFS patients (median age 15.5 years) and 45 healthy controls (median age 15.0 years) were included in this cross-sectional study. Orofacial pain was reported by 26 of 30 JFS patients (86.7%) and by 3 of 45 controls (6.7%; p<0.001). Pain on TMJ palpation was present in 18 of 30 JFS patients (60%) and in 5 of 45 controls (11.1%; p<0.001). Median values of maximum spontaneous mouth opening, voluntary active opening and assisted passive opening were significantly higher in JFS patients than in controls. On multiple regression analysis spontaneous orofacial pain (OR: 21.0; p=0.005), diffuse tenderness on palpation of the masticatory muscles (OR: 14.9; p=0.026) and TMJ hypermobility (OR 1.42; p=0.008) were independently associated with JFS., Conclusions: The high prevalence of TMD in JFS highlights the need for a broader interdisciplinary evaluation of JFS patients. TMJ hypermobility, in addition to orofacial and masticatory muscle pain, is an important clue for the diagnosis of TMD in adolescents with JFS. Elucidating the link between these disorders will advance individualised management and improve treatment efficacy.

A dysfunctional gut microbiota-brain axis is emerging as a potential pathogenic mechanism in epilepsy, particularly in pediatric forms of epilepsy. To add new insights into gut-related changes in acquired epilepsy that develops early in life, we used a multi-omics approach in a rat model with a 56% incidence of epilepsy. The presence of spontaneous seizures was assessed in adult rats (n = 46) 5 months after status epilepticus induced by intra-amygdala kainate at postnatal day 13, by 2 weeks (24/7) ECoG monitoring. Twenty-six rats developed epilepsy (Epi) while the remaining 20 rats (No-Epi) did not show spontaneous seizures. At the end of ECoG monitoring, all rats and their sham controls (n = 20) were sacrificed for quantitative histopathological and immunohistochemical analyses of the gut structure, glia and macrophages, as well as RTqPCR analysis of inflammation/oxidative stress markers. By comparing Epi, No-Epi rats, and sham controls, we found structural, cellular, and molecular alterations reflecting a dysfunctional gut, which were specifically associated with epilepsy. In particular, the villus height-to-crypt depth ratio and number of Goblet cells were reduced in the duodenum of Epi rats vs both No-Epi rats and sham controls (p < 0.01). Villus height and crypt depth in the duodenum and jejunum (p < 0.01) were increased in No-Epi vs both Epi and sham controls. We also detected enhanced Iba1-positive macrophages, together with increased IL1b and NFE2L2 transcripts and TNF protein, in the small intestine of Epi vs both No-Epi and sham control rats (p < 0.01), denoting the presence of inflammation and oxidative stress. Astroglial GFAP-immunostaining was similar in all experimental groups. Metagenomic analysis in the feces collected 5 months after status epilepticus showed that the ratio of two dominant phyla (Bacteroidota-to-Firmicutes) was similarly increased in Epi and No-Epi rats vs sham control rats. Notably, the relative abundance of families, genera, and species associated with SCFA production differed in Epi vs No-Epi rats, describing a bacterial imprint associated with epilepsy. Furthermore, Epi rats showed a blood metabolic signature characterized by changes in lipid metabolism compared to both No-Epi and sham control rats. Our study provides new evidence of long-term gut alterations, along with microbiota-related metabolic changes, occurring specifically in rats that develop epilepsy after brain injury early in life., Competing Interests: Declaration of competing interest The authors have no competing financial interests to declare., (Copyright © 2023. Published by Elsevier Inc.)

Background: Epithelial to mesenchymal transition (EMT) endows cancer cells with pro-metastatic properties, which appear most effective when cells enter an intermediate hybrid (H) state, characterized by integrated mesenchymal (M) and epithelial (E) traits. The reasons for this advantage are poorly known and, especially, it is totally unexplored whether the interplay between H-cells and NK cells could have a role. Here we characterize the pro-metastatic mechanics of non-small cell lung cancer (NSCLC) H-cells and their subset of cancer-initiating cells (CICs), dissecting crucial interactions with NK cells., Methods: Human lung cancer cell lines and sublines representative of E, M, or H states, assessed by proteomics, were analyzed in vivo for their tumor-forming and disseminating capabilities. Interactions with NK cells were investigated in vitro using migration assays, cytotoxic degranulation assays, and evaluation of CD133+ CICs modulation after coculture, and validated in vivo through NK cell neutralization assays. Correlation between EMT status, NK cell infiltration, and survival data, was evaluated in a cohort of surgically resected NSCLC cases (n=79)., Results: We demonstrated that H-cells, have limited dissemination capability but show the highest potential to initiate metastases in vivo. This property was related to their ability to escape NK cell surveillance. Mechanistically, H-cells expressed low levels of NK-attracting chemokines (CXCL1 and CXCL8), generating poorly infiltrated metastases. Accordingly, proteomics and GO enrichment analysis of E, H, M cell lines showed that the related secretory processes could change during EMT.Furthermore, H-CICs uniquely expressed high levels of the inhibitory ligand B7-H3, which protected H-CIC from NK cell-mediated clearance. In vivo neutralization assays confirmed that, indeed, the pro-metastatic properties of H-cells are poorly controlled by NK cells.Finally, the analysis of patients revealed that detection of hybrid phenotypes associated with low NK infiltration in NSCLC clinical specimens could identify a subset of patients with poor prognosis., Conclusions: Our study demonstrates that H-cells play a central role in the metastatic spread in NSCLC. Such pro-metastatic advantage of H-cells is supported by their altered interaction with NK cells and by the critical role of B7-H3 in preserving their H-CIC component, indicating B7-H3 as a potential target in combined NK-based therapies., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Besides recent advances in neonatal care, preterm newborns still develop sex-biased behavioral alterations. Preterms fail to receive placental insulin-like growth factor-1 (IGF-1), a major fetal growth hormone in utero, and low IGF-1 serum levels correlate with preterm poor neurodevelopmental outcomes. Here, we mimicked IGF-1 deficiency of preterm newborns in mice by perinatal administration of an IGF-1 receptor antagonist. This resulted in sex-biased brain microstructural, functional, and behavioral alterations, resembling those of ex-preterm children, which we characterized performing parallel mouse/human behavioral tests. Pharmacological enhancement of GABAergic tonic inhibition by the U.S. Food and Drug Administration-approved drug ganaxolone rescued functional/behavioral alterations in mice. Establishing an unprecedented mouse model of prematurity, our work dissects the mechanisms at the core of abnormal behaviors and identifies a readily translatable therapeutic strategy for preterm brain disorders.

Introduction: Juvenile Sjögren's disease (jSjD) is a rare autoimmune disease characterized by exocrine gland involvement and systemic manifestations, including small vessel vasculitis and Raynaud's phenomenon (RP). We aimed to investigate the microvascular status in jSjD patients by nailfold videocapillaroscopy (NVC) and the potential correlations with clinical and serological features., Methods: Clinical data from thirteen consecutive jSjD patients (11 females and 2 males), with a mean age of 16 ± 4 years, diagnosed before 16 years of age (mean age at diagnosis 12 ± 3) according to the 2016 American College of Rheumatology/EULAR criteria for adult SjD, were collected including age- and sex-matched healthy controls (HCs). Clinical, laboratory, and instrumental data were collected, together with NVC examination. Non-specific and specific NVC parameters were investigated, such as capillary density, capillary dilations, giant capillaries, microhaemorrhages and abnormal shapes. Associations between NVC findings and clinical/serological features were explored and analysed using parametrical and non-parametrical tests., Results: Capillary density reduction correlated significantly with articular involvement (arthralgias) (p = 0.024). Microhaemorrhages correlated with lower C3 levels (p = 0.034). No specific NVC pattern for jSjD was identified, whereas abnormal capillary shapes were significantly higher in jSjD patients than HCs (p = 0.005). NVC abnormalities were not associated with SjD-specific instrumental tests (biopsy, imaging, Schirmer's test). RP was present in 8% of jSjD patients., Conclusions: The reduction of capillary density, as well as microhaemorrhages at NVC analysis, are significantly associated with some clinical aspects like articular involvement and serum biomarkers (C3 reduction). The NVC is suggested as safe and further analysis in jSjD patients., (© 2024. The Author(s).)

Objective: To investigate sleep quality in juvenile fibromyalgia syndrome (JFS) and its effect on the global burden of the disease., Methods: Consecutive patients with JFS who performed full-night polysomnography (PSG) were included in this cross-sectional study. JFS-related symptoms, neuropsychiatric features, and sleep quality were assessed using self-report measures. PSG sleep parameters, including N3 distribution index, were obtained from patients and age-matched healthy controls., Results: We included 25 patients (20 females, median age 15.7 yrs). Nonrestorative sleep was reported by 22 of 25 (88%) patients. Patients with JFS showed significantly longer sleep period time ( P = 0.004) and increased wake after sleep onset ( P = 0.03) compared to healthy peers. The N3 distribution index was significantly lower in patients than in the control group ( P = 0.02). Subjective poor sleep quality was related to Widespread Pain Index (WPI; r s -0.65), Symptom Severity Scale ( r s -0.64), depressive symptoms ( r s -0.58), fatigue ( r s -0.44), and symptom severity upon awakening ( r s -0.65). The N3 distribution index was correlated to depressive symptoms ( r s 0.41) and irritability (r s 0.40). On multiple regression analysis, WPI was predicted by subjective sleep quality (β -0.32, P = 0.04), whereas depressive symptoms were predicted by subjective sleep measures (β -0.32, P = 0.04) and PSG parameters (N3 min: β -0.07, P = 0.03)., Conclusion: Sleep complaints are a key hallmark of JFS and have significant effect on relevant clinical domains of the disease, such as pain and depression., (Copyright © 2023 by the Journal of Rheumatology.)

Natural Killer (NK) cells play a pivotal role in the elimination of tumor cells. The interactions that NK cells can establish with cancer cells in the tumor microenvironment (TME) are crucial for the outcome of the anti-tumor response, possibly resulting in mechanisms able to modulate NK cell effector functions on the one side, and to modify tumor cell phenotype and properties on the other side. This chapter will describe two different experimental approaches for the evaluation of NK-tumor cell interactions. First, a detailed protocol for the setting up of NK-tumor cell co-cultures will be illustrated, followed by information on cell imaging techniques, useful for assessing cell morphology and cytoskeletal changes. The second part will be focused on the description of a proteomic approach aimed at investigating the effect of this crosstalk from another point of view, i.e., characterizing the cellular and molecular pathways modulated in tumor cells following interaction with NK cells. The chapter centers on the interaction between NK and melanoma cells and refers to experimental approaches we set up to study the effects of this cross-talk on the process of the Epithelial-to-Mesenchymal Transition (EMT). Nevertheless, the described protocols can be quite easily adapted to study the interaction of NK cells with adherent tumor cell lines of different origin and histotype, as in our original study, we also analyzed possible NK-induced morphologic changes in the cervix adenocarcinoma HeLa cells and the colon cancer HT29 cells., Competing Interests: Declaration of interests The Authors declare no conflicts of interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Under healthy physiological conditions, living organisms possess a variety of antioxidant mechanisms to scavenge overproduced reactive oxygen species (ROS). However, under pathological circumstances, endogenous antioxidant systems may not be adequate to eliminate the excessive amount of oxidants, and thus, a continuous exogenous antioxidant income is required. In this regard, sumac ( Rhus coriaria ) extract is a good candidate for therapeutic applications, because of its high content of antioxidant polyphenolic compounds. In this work, sumac extract-loaded nanosheets (sumac-nanosheet) have been exploited for loading and controlled release of sumac extract, envisioning topical drug delivery applications. Sumac extract has been obtained through the solvent extraction method, and polymeric nanosheets have been thereafter prepared through the spin coating-assisted layer-by-layer deposition of polycaprolactone (PCL), sumac extract, and poly(d,l-lactic acid) (PDLLA). The collected data show a rich content of the sumac extract in terms of polyphenolic compounds, as well as its strong antioxidant properties. Moreover, for the first time in the literature, we demonstrated the possibility of efficiently loading such extract in polymeric nanosheets and the suitability of this nanoplatform as a reactive oxygen species scavenger in human dermal fibroblasts treated with a pro-oxidant insult.

Aqueous humor (AH) can be easily and safely used to evaluate disease-specific biomarkers in ocular disease. The aim of this study was to identify specific proteins biomarkers in the AH of retinoblastoma (RB) patients at various stages of the disease. We analyzed the proteome of 53 AH samples using high-resolution mass spectrometry. We grouped the samples according to active vitreous seeding (Group 1), active aqueous seeding (Group 2), naive RB (group 3), inactive RB (group 4), and congenital cataracts as the control (Group 5). We found a total of 889 proteins in all samples. Comparative parametric analyses among the different groups revealed three additional proteins expressed in the RB groups that were not expressed in the control group. These were histone H2B type 2-E (HISTH2B2E), InaD-like protein (PATJ), and ubiquitin conjugating enzyme E2 V1 (UBE2V1). Upon processing the data of our study with the OpenTarget Tool software, we found that glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and CD44 were more highly expressed in the RB groups. Our results provide a proteome database regarding AH related to RB disease that may be used as a source of biomarkers. Further prospective studies should validate our finding in a large cohort of RB patients.

Reactive oxygen species (ROS) are a common hallmark of many degenerative diseases, developing in all those cases where a failure of physiological antioxidant mechanisms occurs (in particular, antioxidant enzymes and the glutathione system), or in case of exposure to an extremely high level of oxidants. In this regard, antioxidant natural extracts are promising compounds as preventive or therapeutic agents against ROS-dependent degenerations. In this study, a deep investigation of hazelnut ( Corylus avellana ) extract has been performed in terms of mass spectroscopy, evaluation of phenolic content, and antioxidant capacity. Then, nanostructured lipid carriers (NLCs) have been exploited for encapsulation of the hazelnut extracts in order to achieve prolonged bioactivity, increased stability, and targeting through a sustainable delivery approach. The hazelnut extract-loaded NLCs (NE&#95;NLCs) have been deeply characterized for their stability, production yield, and encapsulation efficiency. Moreover, NE&#95;NLCs showed optimal cytocompatibility on human dermal fibroblast (HDF) cells, as well as excellent antioxidant activity, upon pro-oxidant stimulus on HDF cells., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Emanet, Şen, Pignatelli, Lavarello, Petretto and Ciofani.)

Hereditary periodic recurrent fevers (HRF) are monogenic autoinflammatory associated to mutations of some genes, such as diseases caused by mutations of including MEFV, TNFRSF1A and MVK genes. Despite the identification of the causative genes, the intracellular implications related to each gene variant are still largely unknown. A large -scale proteomic analysis on monocytes of these patients is aimed to identify with an unbiased approach the mean proteins and molecular interaction networks involved in the pathogenesis of these conditions. Monocytes from HRF 15 patients (5 with MFV, 5 TNFRSF1A and 5with MVK gene mutation) and 15 healthy donors (HDs) were analyzed by liquid chromatography and tandem mass spectrometry before and after lipopolysaccharide (LPS) stimulation. Significant proteins were analyzed through a Cytoscape analysis using the ClueGo app to identify molecular interaction networks. Protein networks for each HRF were performed through a STRING database analysis integrated with a DISEAE database query. About 5000 proteins for each HRF were identified. LPS treatment maximizes differences between up-regulated proteins in monocytes of HRF patients and HDs, independently from the disease's activity and ongoing treatments. Proteins significantly modulated in monocytes of the different HRF allowed creating a disease-specific proteomic signatures and interactive protein network. Proteomic analysis is able to dissect the different intracellular pathways involved in the inflammatory response of circulating monocytes in HRF patients. The present data may help to identify a "monocyte proteomic signature" for each condition and unravel new possible unexplored intracellular pathways possibly involved in their pathogenesis. These data will be also useful to identify possible differences and similarities between the different HRFs and some multifactorial recurrent fevers., Competing Interests: FP reports speaker fee from SOBI. MG reports grants and personal fees from Novartis, grants and personal fees from SOBI, outside the submitted work. RP reports speaker fee from SOBI. AB reports speaker fee from SOBI. SV reports speaker fee from SOBI. RC reports speaker fee from SOBI. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Penco, Petretto, Lavarello, Papa, Bertoni, Omenetti, Gueli, Finetti, Caorsi, Volpi and Gattorno.)

Neuroblastoma (NB) is the most common extracranial malignant tumor in children. Although the survival rate of NB has improved over the years, the outcome of NB still remains poor for over 30% of cases. A more accurate risk stratification remains a key point in the study of NB and the availability of novel prognostic biomarkers of "high-risk" at diagnosis could help improving patient stratification and predicting outcome. In this paper we show a biomarker discovery approach applied to the plasma of 172 NB patients. Plasma samples from a first cohort of NB patients and age-matched healthy controls were used for untargeted metabolomics analysis based on high-resolution mass spectrometry (HRMS). Differential expression analysis highlighted a number of metabolites annotated with a high degree of identification. Among them, 3-O-methyldopa (3-O-MD) was validated in a second cohort of NB patients using a targeted metabolite profiling approach and its prognostic potential was also analyzed by survival analysis on patients with 3 years follow-up. High expression of 3-O-MD was associated with worse prognosis in the subset of patients with stage M tumor (log-rank p < 0.05) and, among them, it was confirmed as a prognostic factor able to stratify high-risk patients older than 18 months. 3-O-MD might be thus considered as a novel prognostic biomarker of NB eligible to be included at diagnosis among catecholamine metabolite panels in prospective clinical studies. Further studies are warranted to exploit other potential biomarkers highlighted using our approach., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Barco, Lavarello, Cangelosi, Morini, Eva, Oneto, Uva, Tripodi, Garaventa, Conte, Petretto and Cangemi.)

Parkinson's disease (PD) is a progressive neurodegenerative disease with no satisfactory therapy options. Similar to other neurodegenerative conditions, such as Alzheimer's and Huntington's diseases, oxidative stress plays a key factor in the neurodegeneration process. To counteract the uncontrolled increase of reactive oxygen species (ROS) and oxidative stress-dependent cell death, several preclinical and clinical tests exploit natural-derived organic antioxidants, such as polyphenols. Despite some promising results, free antioxidants show scarce brain accumulation and may exhaust their scavenging activity before reaching the brain. In this work, we developed an antioxidant therapeutic nanoplatform consisting of nano-sized functionalized liposomes loaded with selected polyphenol-rich vegetal extracts with high blood-brain barrier crossing capabilities. The antioxidant extracts were obtained from the grape seeds and skins as a byproduct of wine production ( i.e. , pomace), following a sustainable circular approach with reduced environmental impact. The antioxidant nanoplatform was successfully tested in a relevant in vitro model of PD, where it completely rescued the ROS levels, prevented the aggregation of α-synuclein fibrils, and restored cell viability, paving the way for preclinical translation of the approach.

Glycogen Storage Disease type 1b (GSDIb) is a genetic disorder with long term severe complications. Accumulation of the glucose analog 1,5-anhydroglucitol-6-phosphate (1,5AG6P) in neutrophils inhibits the phosphorylation of glucose in these cells, causing neutropenia and neutrophil dysfunctions. This condition leads to serious infections and inflammatory bowel disease (IBD) in GSDIb patients. We show here that dapagliflozin, an inhibitor of the renal sodium-glucose co-transporter-2 (SGLT2), improves neutrophil function in an inducible mouse model of GSDIb by reducing 1,5AG6P accumulation in myeloid cells., Competing Interests: The authors declare no conflict of interest., (© 2021 Published by Elsevier Inc.)

Liquid-chromatography coupled to high resolution mass spectrometry (LC-HRMS) is currently the method of choice for untargeted metabolomic analysis. The availability of established protocols to achieve a high confidence identification of metabolites is crucial. The aim of this work is to describe the workflow that we have applied to build an Accurate Mass Retention Time (AMRT) database using a commercial metabolite library of standards. LC-HRMS analysis was carried out using a Vanquish Horizon UHPLC system coupled to a Q-Exactive Plus Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Fisher Scientific, Milan, Italy). The fragmentation spectra, obtained with 12 collision energies, were acquired for each metabolite, in both polarities, through flow injection analysis. Several chromatographic conditions were tested to obtain a protocol that yielded stable retention times. The adopted chromatographic protocol included a gradient separation using a reversed phase (Waters Acquity BEH C18) and a HILIC (Waters Acquity BEH Amide) column. An AMRT database of 518 compounds was obtained and tested on real plasma and urine samples analyzed in data-dependent acquisition mode. Our AMRT library allowed a level 1 identification, according to the Metabolomics Standards Initiative, of 132 and 124 metabolites in human pediatric plasma and urine samples, respectively. This library represents a starting point for future metabolomic studies in pediatric settings.

Retinoblastoma (RB) is the most common tumor of the eye in early childhood. Although recent advances in conservative treatment have greatly improved the visual outcome, local tumor control remains difficult in the presence of massive vitreous seeding. Traditional biopsy has long been considered unsafe in RB, due to the risk of extraocular spread. Thus, the identification of new biomarkers is crucial to design safer diagnostic and more effective therapeutic approaches. Exosomes, membrane-derived nanovesicles that are secreted abundantly by aggressive tumor cells and that can be isolated from several biological fluids, represent an interesting alternative for the detection of tumor-associated biomarkers. In this study, we defined the protein signature of exosomes released by RB tumors (RBT) and vitreous seeding (RBVS) primary cell lines by high resolution mass spectrometry. A total of 5666 proteins were identified. Among these, 5223 and 3637 were expressed in exosomes RBT and one RBVS group, respectively. Gene enrichment analysis of exclusively and differentially expressed proteins and network analysis identified in RBVS exosomes upregulated proteins specifically related to invasion and metastasis, such as proteins involved in extracellular matrix (ECM) remodeling and interaction, resistance to anoikis and the metabolism/catabolism of glucose and amino acids.

Leucine-rich repeat kinase 2 (LRRK2) is a large multidomain scaffolding protein with kinase and GTPase activities involved in synaptic vesicle (SV) dynamics. While its role in Parkinson's disease has been largely investigated, little is known about LRRK2 physiological role and until now few proteins have been described as substrates. We have previously demonstrated that LRRK2 through its WD40 domain interacts with synapsin I, an important SV-associated phosphoprotein involved in neuronal development and in the regulation of neurotransmitter release. To test whether synapsin I is substrate for LRRK2 and characterize the properties of its phosphorylation, we used in vitro kinase and binding assays as well as cellular model and site-direct mutagenesis. Using synaptosomes in superfusion, patch-clamp recordings in autaptic WT and synapsin I KO cortical neurons and SypHy assay on primary cortical culture from wild-type and BAC human LRRK2 G2019S mice we characterized the role of LRRK2 kinase activity on glutamate release and SV trafficking. Here we reported that synapsin I is phosphorylated by LRRK2 and demonstrated that the interaction between LRRK2 WD40 domain and synapsin I is crucial for this phosphorylation. Moreover, we showed that LRRK2 phosphorylation of synapsin I at threonine 337 and 339 significantly reduces synapsin I-SV/actin interactions. Using complementary experimental approaches, we demonstrated that LRRK2 controls glutamate release and SV dynamics in a kinase activity and synapsin I-dependent manner. Our findings show that synapsin I is a LRRK2 substrate and describe a novel mechanisms of regulation of glutamate release by LRRK2 kinase activity., (© 2019 International Society for Neurochemistry.)

NETs constitute a network of DNA and proteins released by neutrophils in response to infectious and immunologic triggers. NET proteins are recognized as autoantigens in ANCA vasculitis; limited knowledge is available in other autoimmune pathologies. The composition of NETs produced ex vivo by resting and Phorbol-myristate acetate (PMA) stimulated neutrophils was analyzed by high-throughput Fusion Orbitrap technology in 16 patients with Systemic Lupus Erythematosus/Lupus nephritis (9 SLE/7 LN) and in 11 controls. Seven-hundred proteins were characterized and specific fingerprints discriminated LN from SLE. We focused on methyl-oxidized αenolase (methionine sulfoxide 93) that was markedly increased in NETs from LN and was localized in NET filaments in tight connection and outlying DNA. The isotype of anti-αenolase antibodies was IgG2 in LN and IgG4 in other autoimmune glomerulonephritis (Membranous Nephropathy, MN); serum anti-αenolase IgG2 were higher in LN than in SLE and absent in MN. The same IgG2 antibodies recognized 5 epitopes of the protein one containing methionine sulphoxide 93. In conclusion, specific NET protein fingerprints characterize different subsets of SLE; methyl-oxidized αenolase is over-expressed in LN. Circulating anti-αenolase IgG2 recognize the oxidized epitope and are high in serum of LN patients. Post-translational modified NET proteins contribute to autoimmunity in patients with LN.

Background: Prednisone (PDN) in juvenile dermatomyositis (JDM), alone or in association with other immunosuppressive drugs, namely methotrexate (MTX) and cyclosporine (CSA), represents the first-line treatment option for new onset JDM patients. No clear evidence based guidelines are actually available to standardize the tapering and discontinuation of glucocorticoids (GC) in JDM. Aim of our study was to provide an evidence-based proposal for GC tapering/discontinuation in new onset juvenile dermatomyositis (JDM), and to identify predictors of clinical remission and GC discontinuation., Methods: New onset JDM children were randomized to receive either PDN alone or in combination with methotrexate (MTX) or cyclosporine (CSA). In order to derive steroid tapering indications, PRINTO/ACR/EULAR JDM core set measures (CSM) and their median absolute and relative percent changes over time were compared in 3 groups. Group 1 included those in clinical remission who discontinued PDN, with no major therapeutic changes (MTC) (reference group) and was compared with those who did not achieve clinical remission, without or with MTC (Group 2 and 3, respectively). A logistic regression model identified predictors of clinical remission with PDN discontinuation., Results: Based on the median change in the CSM of 30/139 children in Group 1, after 3 pulses of methyl-prednisolone, GC could be tapered from 2 to 1 mg/kg/day in the first two months from onset if any of the CSM decreased by 50-94%, and from 1 to 0.2 mg/kg/day in the following 4 months if any CSM further decreased by 8-68%, followed by discontinuation in the ensuing 18 months. The achievement of PRINTO JDM 50-70-90 response after 2 months of treatment (ORs range 4.5-6.9), an age at onset > 9 years (OR 4.6) and the combination therapy PDN + MTX (OR 3.6) increase the probability of achieving clinical remission (p < 0.05)., Conclusions: This is the first evidence-based proposal for glucocorticoid tapering/discontinuation based on the change in JDM CSM of disease activity., Trial Registration: Trial full title: Five-Year Single-Blind, Phase III Effectiveness Randomized Actively Controlled Clinical Trial in New Onset Juvenile Dermatomyositis: Prednisone versus Prednisone plus Cyclosporine A versus Prednisone plus Methotrexate. EUDRACT registration number: 2005-003956-37 ., Clinical Trial: gov is NCT00323960 . Registered on 17 August 2005.

Tumor cell plasticity is a major obstacle for the cure of malignancies as it makes tumor cells highly adaptable to microenvironmental changes, enables their phenotype switching among different forms, and favors the generation of prometastatic tumor cell subsets. Phenotype switching toward more aggressive forms involves different functional, phenotypic, and morphologic changes, which are often related to the process known as epithelial-mesenchymal transition (EMT). In this study, we report natural killer (NK) cells may increase the malignancy of melanoma cells by inducing changes relevant to EMT and, more broadly, to phenotype switching from proliferative to invasive forms. In coculture, NK cells induced effects on tumor cells similar to those induced by EMT-promoting cytokines, including upregulation of stemness and EMT markers, morphologic transition, inhibition of proliferation, and increased capacity for Matrigel invasion. Most changes were dependent on the engagement of NKp30 or NKG2D and the release of cytokines including IFNγ and TNFα. Moreover, EMT induction also favored escape from NK-cell attack. Melanoma cells undergoing EMT either increased NK-protective HLA-I expression on their surface or downregulated several tumor-recognizing activating receptors on NK cells. Mass spectrometry-based proteomic analysis revealed in two different melanoma cell lines a partial overlap between proteomic profiles induced by NK cells or by EMT cytokines, indicating that various processes or pathways related to tumor progression are induced by exposure to NK cells. Significance: NK cells can induce prometastatic properties on melanoma cells that escape from killing, providing important clues to improve the efficacy of NK cells in innovative antitumor therapies. Cancer Res; 78(14); 3913-25. ©2018 AACR ., (©2018 American Association for Cancer Research.)

The release of soluble ligands of activating Natural Killer (NK) cell receptors may represent a regulatory mechanism of NK cell function both in physiologic and in pathologic conditions. Here, we identified the extracellular matrix protein Nidogen-1 (NID1) as a ligand of NKp44, an important activating receptor expressed by activated NK cells. When released as soluble molecule, NID1 regulates NK cell function by modulating NKp44-induced IFN-γ production or cytotoxicity. In particular, it also modulates IFN-γ production induced by Platelet-Derived Growth Factor (PDGF)-DD following NKp44 engagement. We also show that NID1 may be present at the cell surface. In this form or when bound to a solid support (bNID1), NID1 fails to induce NK cell cytotoxicity or cytokine release. However, analysis by mass spectrometry revealed that exposure to bNID1 can induce in human NK cells relevant changes in the proteomic profiles suggesting an effect on different biological processes.

Chimeric antigen receptor (CAR) T-cell therapy has been shown to be dramatically effective in the treatment of B-cell malignancies. However, there are still substantial obstacles to overcome, before similar responses can be achieved in patients with solid tumors. We evaluated both in vitro and in a preclinical murine model the efficacy of different 2nd and 3rd generation CAR constructs targeting GD2, a disial-ganglioside expressed on the surface of neuroblastoma (NB) tumor cells. In order to address potential safety concerns regarding clinical application, an inducible safety switch, namely inducible Caspase-9 (iC9), was also included in the vector constructs. Our data indicate that a 3rd generation CAR incorporating CD28.4-1BB costimulatory domains is associated with improved anti-tumor efficacy as compared with a CAR incorporating the combination of CD28.OX40 domains. We demonstrate that the choice of 4-1BB signaling results into significant amelioration of several CAR T-cell characteristics, including: 1) T-cell exhaustion, 2) basal T-cell activation, 3) in vivo tumor control and 4) T-cell persistence. The fine-tuning of T-cell culture conditions obtained using IL7 and IL15 was found to be synergic with the CAR.GD2 design in increasing the anti-tumor activity of CAR T cells. We also demonstrate that activation of the suicide gene iC9, included in our construct without significantly impairing neither CAR expression nor anti-tumor activity, leads to a prompt induction of apoptosis of GD2.CAR T cells. Altogether, these findings are instrumental in optimizing the function of CAR T-cell products to be employed in the treatment of children with NB.

Neuroblastoma (NB) is the most common extracranial pediatric solid tumor. Around 70% of patients with metastatic disease at diagnosis present bone-marrow infiltration, which is considered a marker of poor outcome; however, the mechanism underlying this specific tropism has to be elucidated. Tumor-derived exosomes may support metastatic progression in several tumors by interacting with the microenvironment, and may serve as tumor biomarkers. The main objective of this study is to identify an exosomal signature associated with NB metastatic bone-marrow dissemination. Therefore, the proteomic cargo of exosomes isolated from NB cell lines derived from primary tumor and bone-marrow metastasis is characterized. The comparison among exosomal proteins show 15 proteins exclusively present in primary tumor-derived exosomes, mainly involved in neuronal development, and 6 proteins in metastasis-derived exosomes related to cancer progression. Significant proteins obtain with statistical analysis performed between the two groups, reveal that primary tumor exosomes contain a higher level of proteins involved in extra-cellular matrix (ECM) assembly and adhesion, as well as in neuronal development. Exosomes isolated from bone-marrow metastasis exhibit proteins involved in ameboidal cell migration and mitochondrial activity. This work suggests that proteomic profiling of NB-derived exosomes reflects the tumor stage and may be considered as potential tumor biomarker., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

IL-27, a member of the IL-12-family of cytokines, has shown anti-tumor activity in several pre-clinical models due to anti-proliferative, anti-angiogenic and immune-enhancing effects. On the other hand, IL-27 demonstrated immune regulatory activities and inhibition of auto-immunity in mouse models. Also, we reported that IL-27, similar to IFN-γ, induces the expression of IL-18BP, IDO and PD-L1 immune regulatory molecules in human cancer cells. Here, a proteomic analysis reveals that IL-27 and IFN-γ display a broad overlap of functions on human ovarian cancer cells. Indeed, among 990 proteins modulated by either cytokine treatment in SKOV3 cells, 814 showed a concordant modulation by both cytokines, while a smaller number (176) were differentially modulated. The most up-regulated proteins were common to both IFN-γ and IL-27. In addition, functional analysis of IL-27-regulated protein networks highlighted pathways of interferon signaling and regulation, antigen presentation, protection from natural killer cell-mediated cytotoxicity, regulation of protein polyubiquitination and proteasome, aminoacid catabolism and regulation of viral protein levels.Importantly, we found that IL-27 induced HLA class I molecule expression in human cancer cells of different histotypes, including tumor cells showing very low expression. IL-27 failed only in a cancer cell line bearing a homozygous deletion in the B2M gene. Altogether, these data point out to a broad set of activities shared by IL-27 and IFN-γ, which are dependent on the common activation of the STAT1 pathway. These data add further explanation to the anti-tumor activity of IL-27 and also to its dual role in immune regulation.

We demonstrated that γδ T cells of patients given HLA-haploidentical HSCT after removal of αβ + T cells and CD19 + B cells are endowed with the capacity of killing leukemia cells after ex vivo treatment with zoledronic acid (ZOL). Thus, we tested the hypothesis that infusion of ZOL in patients receiving this type of graft may enhance γδ T-cell cytotoxic activity against leukemia cells. ZOL was infused every 28 d in 43 patients; most were treated at least twice. γδ T cells before and after ZOL treatments were studied in 33 of these 43 patients, till at least 7 mo after HSCT by high-resolution mass spectrometry, flow-cytometry, and degranulation assay. An induction of Vδ2-cell differentiation, paralleled by increased cytotoxicity of both Vδ1 and Vδ2 cells against primary leukemia blasts was associated with ZOL treatment. Cytotoxic activity was further increased in Vδ2 cells, but not in Vδ1 lymphocytes in those patients given more than one treatment. Proteomic analysis of γδ T cells purified from patients showed upregulation of proteins involved in activation processes and immune response, paralleled by downregulation of proteins involved in proliferation. Moreover, a proteomic signature was identified for each ZOL treatment. Patients given three or more ZOL infusions had a better probability of survival in comparison to those given one or two treatments (86% vs. 54%, respectively, p = 0.008). Our data indicate that ZOL infusion in pediatric recipients of αβ T- and B-cell-depleted HLA-haploidentical HSCT promotes γδ T-cell differentiation and cytotoxicity and may influence the outcome of patients.

Exosomes are 40-100-nm vesicles released by most cell types after fusion of multivesicular endosomes with the plasma membrane. Exosomes, ubiquitary in body fluids including urines, contain proteins and RNA species specific of the tissue of origin. Exosomes from urine have been extensively studied as a promising reservoir for disease biomarkers. Here, we report the proteome analysis of urinary exosomes compared to urinoma, studied by Orbitrap mass spectrometry. A discovery approach was utilized on the sample. 3429 proteins were present, with minimal overlapping among exosome and urinoma. 959 proteins (28%) in exosome and 1478 proteins (43%) in urinoma were exclusively present in only one group. By cytoscape analysis, the biological process gene ontology was correlated to their probability (P ≤ 0.05) to be functional. This was never studied before and showed a significant clustering around metabolic functions, in particular to aerobic ATP production. Urinary exosomes carry out oxidative phosphorylation, being able to synthesize ATP and consume oxygen. A previously unsuspected function emerges for human urinary exosomes as bioactive vesicles that consume oxygen to aerobically synthesize ATP. Determination of normal human urine proteome can help generate the healthy urinary protein database for comparison, useful for various renal diseases., Biological Significance: The findings reported represent a significant advance in the understanding of the healthy human urinary proteome. The methodology utilized to analyze the collection of proteomic data allowed the assessment of the unique composition of urinary exosomes with respect to urinoma and to elucidate the presence in the former of molecular pathways previously unknown. The paper has the potential to impact its field of research, due to the biological relevance of the metabolic capacity of urinary exosomes, which may represent their important general feature., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Exosomes are nanovesicles, derived from the endocytic pathway, released by most cell types and found in many body fluids, including urine. A variety of exosomal functions have been reported, including transfer of RNA, cell communication, control of apoptosis and protein lifespan. Exosomes from mesenchymal stem cells can rescue bioenergetics of injured cells. Here the urinary exosome proteome, non-urinary exosome proteome and urinome are compared. A consistent number of identified proteins cluster to metabolic functions. Cytoscape software analysis based on biological processes gene ontology database shows that metabolic pathways such as aerobic glycolysis and oxidative phosphorylation have a high probability (p ≤ 0.05) of being expressed and therefore functional. A metabolic function appears to be associated with human urinary exosomes, whose relevance experimental studies can assess.

Tumor chemoresistance is associated with high aerobic glycolysis rates and reduced oxidative phosphorylation, a phenomenon called "Warburg effect" whose reversal could impair the ability of a wide range of cancer cells to survive in the presence or absence of chemotherapy. In previous studies, Short-term-starvation (STS) was shown to protect normal cells and organs but to sensitize different cancer cell types to chemotherapy but the mechanisms responsible for these effects are poorly understood. We tested the cytotoxicity of Oxaliplatin (OXP) combined with a 48hour STS on the progression of CT26 colorectal tumors. STS potentiated the effects of OXP on the suppression of colon carcinoma growth and glucose uptake in both in vitro and in vivo models. In CT26 cells, STS down-regulated aerobic glycolysis, and glutaminolysis, while increasing oxidative phosphorylation. The STS-dependent increase in both Complex I and Complex II-dependent O(2) consumption was associated with increased oxidative stress and reduced ATP synthesis. Chemotherapy caused additional toxicity, which was associated with increased succinate/Complex II-dependent O(2) consumption, elevated oxidative stress and apoptosis .These findings indicate that the glucose and amino acid deficiency conditions imposed by STS promote an anti-Warburg effect characterized by increased oxygen consumption but failure to generate ATP, resulting in oxidative damage and apoptosis.

Combinatorial peptide ligand libraries (CPLLs) tend to bind complex molecules such as dyes due to their aromatic, heterocyclic, hydrophobic, and ionic nature that may affect the protein capture specificity. In this experimental work Alcian Blue 8GX, a positively charged phthalocyanine dye well-known to bind to glycoproteins and to glucosaminoglycans, was adsorbed on a chemically modified CPLL solid phase, and the behavior of the resulting conjugate was then investigated. The control and dye-adsorbed beads were used to harvest the human urinary proteome at physiological pH, this resulting in a grand total of 1151 gene products identified after the capture. Although the Alcian Blue-modified CPLL incremented the total protein capture by 115 species, it particularly enriched some families among the harvested proteins, such as glycoproteins and nucleotide-binding proteins. This study teaches that it is possible, via the two combined harvest mechanisms, to drive the CPLL capture toward the enrichment of specific protein categories.

It is currently unknown how many proteins can be detected in urine. Improving the analytical approach would increase their number and potentially strengthen their predictive potential in diseases. We developed a combination of analytical procedures for maximizing sensitivity and reproducibility of normal human urinary proteome analysis based on ultracentrifugation, vesicles separation, combinatorial peptide ligand libraries (CPLL) and solvent removal of pigments. Proteins were identified by an Orbitrap Velos Mass Spectrometry. Overall, 3429 proteins were characterized: most components (1615) were contained in vesicles while the remaining 1794 were equally distributed among CPLL and butanol insoluble fractions. Several proteins were detected exclusively in one of the phases of the procedure, suggesting that each step is crucial in the fractionation strategy. Many (1724) proteins are described here whose presence in urine has never been reported and represents a potential source of information considering that urine is the unique site of excretion of products of interaction of metabolic processes. Improving the characterization of normal urinary proteome would also represent the basis for the analysis of urine biomarkers in human diseases., Biological Significance: Sub-fractionating normal urine by successive steps (vesicle separation, CPLL and solvent treatments) allowed the identification of 3429 proteins, a relevant part (1724) being detected for the first time in urine. Several proteins of new description have been implicated in physiology pathways and in pathologies thus representing a potential source of new information on both metabolic processes and diseases., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The limits on protein detection in urine are unknown. Improving the analytical approach to detection would increase the number of identified proteins and potentially strengthen their predictive potential in diseases. Here, we present the data that resulted from a combination of analytical procedures for maximizing sensitivity and reproducibility of normal human urinary proteome analysis. These procedures are ultracentrifugation, vesicle separation, combinatorial peptide ligand libraries (CPLL) and solvent removal of pigments. Proteins were identified by an Orbitrap Velos Mass Spectrometry. 3429 proteins are characterized, 1724 of which are novel discoveries. The data are related to Santucci et al. (in press) [1] and available both here and at ChorusProject.org under project name "From hundreds to thousands: widening the normal human Urinome". The material supplied to Chorus Progect.org includes technical MS spectra data only.

The present report tries to assess the possibility of performing capture of proteomes via combinatorial peptide ligand libraries (CPLL) in a two-dimensional (2D) mode, i.e. via orthogonal complementarity in the capture phase. To that aim, serum proteins are captured at physiological pH either at low ionic strength (25mM NaCl) or at high concentrations of lyotropic salts of the Hofmeister series (1M ammonium sulphate) favouring hydrophobic interaction. Indeed such 2D mechanisms seems to be operative, since 52% of the captured proteins are common to the two capture modes, 20% are specific only of the "ionic" interaction mode and 28% are found only in the "hydrophobically" driven interaction. As an additional bonus, losses of protein species from the initial sample, one of the major drawbacks of CPLLs, are diminished to about 5% and are found only in the ionic capture, whereas the hydrophobically engendered capture is loss-free., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Background Context: Acute low back pain (LBP) is a frequent cause of physician visits and sick leave. Patients with longer sick leave account for most costs associated with LBP. Most research on risk factors for prolonged sick leave because of LBP has been done in Anglo-Saxon or Northern European populations with occupational LBP. Few studies have been conducted in less affluent Latin countries., Purpose: To investigate the prevalence of acute LBP as a cause of sick leave and the variables associated with longer work absence (WA) because of acute LBP in Chile., Study Design: A retrospective study of nonoccupational sick leave certificates issued in a 1-year period by the Chilean Public Health System., Patient Sample: Ten thousand cases with nonoccupational sick leave certificates issued with the diagnosis of acute LBP., Outcome Measures: Prevalence of acute LBP as a cause of sick leave, days of WA, and subsidy payment., Methods: A review of sick leave certificates from patients with different diagnoses was done to determine the prevalence of acute LBP as a cause of sick leave. We investigated whether age, gender, history of at least one episode of sick leave in the previous year because of acute LBP or because of other diagnoses, stipendiary subsidy paid (converted to US $), work activity, occupation, and the specialty of the attending physician influenced the length of sick leave., Results: Acute LBP represented 5.4% of cases causing sick leave. Patients with acute LBP who had significantly longer sick leave than the rest of the population were patients with an episode of WA because of LBP in the previous year (14% longer sick leave than patients without that history), manual workers (35% longer than nonmanual workers), and patients seen by orthopedic surgeons (43% longer than patients seen by other medical specialists). Nonpregnant women and men had similar durations of sick leave because of acute LBP., Conclusions: Physicians who treat patients with LBP should be aware of these variables associated with longer sick leave because of acute LBP, which represents a significant burden to any society and is an important epidemiologic problem.

The biophysical profile is an excellent test for assessment of the fetoplacental unit, for analyze is value in twin gestation, we study prospectively 81 multiple gestations. The fetuses were examined weekly antepartum. We use the modified profile of Manning which include only the sonographic criteria: the figures for predict stillborn were: sensitivity of 66.7%, specificity of 98.8%, positive predictive value of 50% and negative predictive value of 99.4% and predict between distressed and healthy fetus of a pair; 94.6% of fetus perform good biophysical profile score twenty minutes. We conclude that the fetal biophysical profile is the elective tool in the assessment of twin gestations.