CD99 Modulates the Proteomic Landscape of Ewing Sarcoma Cells and Related Extracellular Vesicles.

Ewing sarcoma (EWS) is an aggressive pediatric bone tumor characterized by unmet clinical needs and an incompletely understood epigenetic heterogeneity. Here, we considered CD99, a major surface molecule hallmark of EWS malignancy. Fluctuations in CD99 expression strongly impair cell dissemination, differentiation, and death. CD99 is also loaded within extracellular vesicles (EVs), and the delivery of CD99-positive or CD99-negative EVs dynamically exerts oncogenic or oncosuppressive functions to recipient cells, respectively. We undertook mass spectrometry and functional annotation analysis to investigate the consequences of CD99 silencing on the proteomic landscape of EWS cells and related EVs. Our data demonstrate that (i) the decrease in CD99 leads to major changes in the proteomic profile of EWS cells and EVs; (ii) intracellular and extracellular compartments display two distinct signatures of differentially expressed proteins; (iii) proteomic changes converge to the modulation of cell migration and immune-modulation biological processes; and (iv) CD99-silenced cells and related EVs are characterized by a migration-suppressive, pro-immunostimulatory proteomic profile. Overall, our data provide a novel source of CD99-associated protein biomarkers to be considered for further validation as mediators of EWS malignancy and as EWS disease liquid biopsy markers.

Astroglial calcium signaling and homeostasis in tuberous sclerosis complex.

Tuberous Sclerosis Complex (TSC) is a multisystem genetic disorder characterized by the development of benign tumors in various organs, including the brain, and is often accompanied by epilepsy, neurodevelopmental comorbidities including intellectual disability and autism. A key hallmark of TSC is the hyperactivation of the mechanistic target of rapamycin (mTOR) signaling pathway, which induces alterations in cortical development and metabolic processes in astrocytes, among other cellular functions. These changes could modulate seizure susceptibility, contributing to the progression of epilepsy and its associated comorbidities. Epilepsy is characterized by dysregulation of calcium (Ca2+) channels and intracellular Ca2+ dynamics. These factors contribute to hyperexcitability, disrupted synaptogenesis, and altered synchronization of neuronal networks, all of which contribute to seizure activity. This study investigates the intricate interplay between altered Ca2+ dynamics, mTOR pathway dysregulation, and cellular metabolism in astrocytes. The transcriptional profile of TSC patients revealed significant alterations in pathways associated with cellular respiration, ER and mitochondria, and Ca2+ regulation. TSC astrocytes exhibited lack of responsiveness to various stimuli, compromised oxygen consumption rate and reserve respiratory capacity underscoring their reduced capacity to react to environmental changes or cellular stress. Furthermore, our study revealed significant reduction of store operated calcium entry (SOCE) along with strong decrease of basal mitochondrial Ca2+ concentration and Ca2+ influx in TSC astrocytes. In addition, we observed alteration in mitochondrial membrane potential, characterized by increased depolarization in TSC astrocytes. Lastly, we provide initial evidence of structural abnormalities in mitochondria within TSC patient-derived astrocytes, suggesting a potential link between disrupted Ca2+ signaling and mitochondrial dysfunction. Our findings underscore the complexity of the relationship between Ca2+ signaling, mitochondria dynamics, apoptosis, and mTOR hyperactivation. Further exploration is required to shed light on the pathophysiology of TSC and on TSC associated neuropsychiatric disorders offering further potential avenues for therapeutic development.

Novel cellular systems unveil mucosal melanoma initiating cells and a role for PI3K/Akt/mTOR pathway in mucosal melanoma fitness.

BACKGROUND: Mucosal Melanomas (MM) are highly aggressive neoplasms arising from mucosal melanocytes. Current treatments offer a limited survival benefit for patients with advanced MM; moreover, the lack of pre-clinical cellular systems has significantly limited the understanding of their immunobiology. METHODS: Five novel cell lines were obtained from patient-derived biopsies of MM arising in the sino-nasal mucosa and designated as SN-MM1-5. The morphology, ultrastructure and melanocytic identity of SN-MM cell lines were validated by transmission electron microscopy and immunohistochemistry. Moreover, in vivo tumorigenicity of SN-MM1-5 was tested by subcutaneous injection in NOD/SCID mice. Molecular characterization of SN-MM cell lines was performed by a mass-spectrometry proteomic approach, and their sensitivity to PI3K chemical inhibitor LY294002 was validated by Akt activation, measured by pAkt(Ser473) and pAkt(Thr308) in immunoblots, and MTS assay. RESULTS: This study reports the validation and functional characterization of five newly generated SN-MM cell lines. Compared to the normal counterpart, the proteomic profile of SN-MM is consistent with transformed melanocytes showing a heterogeneous degree of melanocytic differentiation and activation of cancer-related pathways. All SN-MM cell lines resulted tumorigenic in vivo and display recurrent structural variants according to aCGH analysis. Of relevance, the microscopic analysis of the corresponding xenotransplants allowed the identification of clusters of MITF-/CDH1-/CDH2 + /ZEB1 + /CD271 + cells, supporting the existence of melanoma-initiating cells also in MM, as confirmed in clinical samples. In vitro, SN-MM cell lines were sensitive to cisplatin, but not to temozolomide. Moreover, the proteomic analysis of SN-MM cell lines revealed that RICTOR, a subunit of mTORC2 complex, is the most significantly activated upstream regulator, suggesting a relevant role for the PI3K-Akt-mTOR pathway in these neoplasms. Consistently, phosphorylation of NDRG1 and Akt activation was observed in SN-MM, the latter being constitutive and sustained by PTEN loss in SN-MM2 and SN-MM3. The cell viability impairment induced by LY294002 confirmed a functional role for the PI3K-Akt-mTOR pathway in SN-MM cell lines. CONCLUSIONS: Overall, these novel and unique cellular systems represent relevant experimental tools for a better understanding of the biology of these neoplasms and, as an extension, to MM from other sites.

The Pro-Oncogenic Sphingolipid-Metabolizing Enzyme ß-Galactosylceramidase Modulates the Proteomic Landscape in BRAF(V600E)-Mutated Human Melanoma Cells.

ß-Galactosylceramidase (GALC) is a lysosomal enzyme involved in sphingolipid metabolism by removing ß-galactosyl moieties from ß-galactosylceramide and ß-galactosylsphingosine. Previous observations have shown that GALC may exert pro-oncogenic functions in melanoma and Galc silencing, leading to decreased oncogenic activity in murine B16 melanoma cells. The tumor-driving BRAF(V600E) mutation is present in approximately 50% of human melanomas and represents a major therapeutic target. However, such mutation is missing in melanoma B16 cells. Thus, to assess the impact of GALC in human melanoma in a more relevant BRAF-mutated background, we investigated the effect of GALC overexpression on the proteomic landscape of A2058 and A375 human melanoma cells harboring the BRAF(V600E) mutation. The results obtained by liquid chromatography-tandem mass spectrometry (LC-MS/MS) demonstrate that significant differences exist in the protein landscape expressed under identical cell culture conditions by A2058 and A375 human melanoma cells, both harboring the same BRAF(V600E)-activating mutation. GALC overexpression resulted in a stronger impact on the proteomic profile of A375 cells when compared to A2058 cells (261 upregulated and 184 downregulated proteins versus 36 and 14 proteins for the two cell types, respectively). Among them, 25 proteins appeared to be upregulated in both A2058-upGALC and A375-upGALC cells, whereas two proteins were significantly downregulated in both GALC-overexpressing cell types. These proteins appear to be involved in melanoma biology, tumor invasion and metastatic dissemination, tumor immune escape, mitochondrial antioxidant activity, endoplasmic reticulum stress responses, autophagy, and/or apoptosis. Notably, analysis of the expression of the corresponding genes in human skin cutaneous melanoma samples (TCGA, Firehose Legacy) using the cBioPortal for Cancer Genomics platform demonstrated a positive correlation between GALC expression and the expression levels of 14 out of the 27 genes investigated, thus supporting the proteomic findings. Overall, these data indicate for the first time that the expression of the lysosomal sphingolipid-metabolizing enzyme GALC may exert a pro-oncogenic impact on the proteomic landscape in BRAF-mutated human melanoma.

In-Depth Proteomic Analysis of Blood Circulating Small Extracellular Vesicles.

Circulating small extracellular vesicles (sEVs), also called exosomes, are key players in the investigation of cell-cell communication mechanisms and in the identification of new potential biomarkers. These particles can carry proteins, DNA, mRNA, miRNA, lipids and metabolites that are transported all over the human body, potentially reaching all the cells. In particular, proteins, which are well-known biological actors in cell signalling, will be discussed in this context. In this article, we present a mass spectrometry approach for the in-depth characterization of the sEVs proteome. The protocols include strategies for the isolation and purification of sEVs, for the extraction of proteins and the purification of sEVs proteins by the immunodepletion of the most abundant plasmatic proteins. Finally, bioinformatic analysis for the extraction of the most important biological features associated with the proteomic content of sEVs is reported.

The Need for Biomarkers in the ALS-FTD Spectrum: A Clinical Point of View on the Role of Proteomics.

Frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are severely debilitating and progressive neurodegenerative disorders. A distinctive pathological feature of several neurodegenerative diseases, including ALS and FTD, is the deposition of aberrant protein inclusions in neuronal cells, which leads to cellular dysfunction and neuronal damage and loss. Despite this, to date, the biological process behind developing these protein inclusions must be better clarified, making the development of disease-modifying treatment impossible until this is done. Proteomics is a powerful tool to characterize the expression, structure, functions, interactions, and modifications of proteins of tissue and biological fluid, including plasma, serum, and cerebrospinal fluid. This protein-profiling characterization aims to identify disease-specific protein alteration or specific pathology-based mechanisms which may be used as markers of these conditions. Our narrative review aims to highlight the need for biomarkers and the potential use of proteomics in clinical practice for ALS-FTD spectrum disorders, considering the emerging rationale in proteomics for new drug development. Certainly, new data will emerge in the near future in this regard and support clinicians in the development of personalized medicine.

Local Allergic Rhinitis: Lights and Shadows of a Mysterious Entity.

Local allergic rhinitis (LAR) is, to date, a debated and complex entity, still orphan of global consideration and a multicentric approach. LAR does not seem to find a proper positioning in the classic classifications and phenotypes of chronic rhinitis, and its pathophysiology relies specifically on the presence of local IgE. These patients in fact have a suggestive clinical history of allergic rhinitis in the presence of negative skin prick tests and serum IgE tests for the suspect allergen. Nasal allergen challenge, assessment of local IgE, basophil activation test (BAT), and nasal cytology are, at the moment, the most used tests in the diagnostic approach to the disease, despite their limitations. Considering that the correct interpretation of diagnostic tests and their clinical relevance is fundamental in the assessment of the right diagnosis and the subsequent therapy, we propose a new diagnostic approach that encompasses all of these methodologies and suggest that several pragmatic randomized control trials as well as prospective, multicentric studies directed at the long-term follow-up of LAR be carried out to further investigate this debated entity.

Tumor Suppressor Role of Wild-Type P53-Dependent Secretome and Its Proteomic Identification in PDAC.

The study of the cancer secretome is gaining even more importance in cancers such as pancreatic ductal adenocarcinoma (PDAC), whose lack of recognizable symptoms and early detection assays make this type of cancer highly lethal. The wild-type p53 protein, frequently mutated in PDAC, prevents tumorigenesis by regulating a plethora of signaling pathways. The importance of the p53 tumor suppressive activity is not only primarily involved within cells to limit tumor cell proliferation but also in the extracellular space. Thus, loss of p53 has a profound impact on the secretome composition of cancer cells and marks the transition to invasiveness. Here, we demonstrate the tumor suppressive role of wild-type p53 on cancer cell secretome, showing the anti-proliferative, apoptotic and chemosensitivity effects of wild-type p53 driven conditioned medium. By using high-resolution SWATH-MS technology, we characterized the secretomes of p53-deficient and p53-expressing PDAC cells. We found a great number of secreted proteins that have known roles in cancer-related processes, 30 of which showed enhanced and 17 reduced secretion in response to p53 silencing. These results are important to advance our understanding on the link between wt-p53 and cancer microenvironment. In conclusion, this approach may detect a secreted signature specifically driven by wild-type p53 in PDAC.

Thermal processing of insect allergens and IgE cross-recognition in Italian patients allergic to shrimp, house dust mite and mealworm.

Edible insects are considered as a promising and sustainable alternative protein source for humans, although risk assessments, with particular reference to the allergic potential of insect proteins, are required. Considering that insects are likely to be consumed after processing, it is crucial to assess how processing can influence allergenicity. In our study, we investigated how boiling and frying affect the IgE cross-recognition of proteins from five edible insects (mealworm, buffalo worm, silkworm, cricket and grasshopper). We considered three groups of Italian patients allergic to shrimps and to house dust mites, who had never consumed insects before and two subjects with occupational allergy and food sensitization to mealworm. Our data suggest that thermal processing may change the solubility of proteins, thereby resulting in a protein shift from water-soluble fractions to water-insoluble fractions. Immunoblot and LC-MS/MS analyses have shown that tropomyosin may play an important role as a cross-allergen for house dust mite and shrimp allergic patients, while larval cuticle protein seems to play a major role in the cross-reactivity of patients primarily sensitized to mealworm. On the basis of our results, the effects of processing appear to be protein-, species- and treatment-specific. Therefore, house dust mite, shrimp and mealworm allergic patients should consume insects with caution, even after thermal processing.

Circulating Exosomes Are Strongly Involved in SARS-CoV-2 Infection.

Knowledge of the host response to the novel coronavirus SARS-CoV-2 remains limited, hindering the understanding of COVID-19 pathogenesis and the development of therapeutic strategies. During the course of a viral infection, host cells release exosomes and other extracellular vesicles carrying viral and host components that can modulate the immune response. The present study used a shotgun proteomic approach to map the host circulating exosomes' response to SARS-CoV-2 infection. We investigated how SARS-CoV-2 infection modulates exosome content, exosomes' involvement in disease progression, and the potential use of plasma exosomes as biomarkers of disease severity. A proteomic analysis of patient-derived exosomes identified several molecules involved in the immune response, inflammation, and activation of the coagulation and complement pathways, which are the main mechanisms of COVID-19-associated tissue damage and multiple organ dysfunctions. In addition, several potential biomarkers-such as fibrinogen, fibronectin, complement C1r subcomponent and serum amyloid P-component-were shown to have a diagnostic feature presenting an area under the curve (AUC) of almost 1. Proteins correlating with disease severity were also detected. Moreover, for the first time, we identified the presence of SARS-CoV-2 RNA in the exosomal cargo, which suggests that the virus might use the endocytosis route to spread infection. Our findings indicate circulating exosomes' significant contribution to several processes-such as inflammation, coagulation, and immunomodulation-during SARS-CoV-2 infection. The study's data are available via ProteomeXchange with the identifier PXD021144.

Immunostimulants in respiratory diseases: focus on Pidotimod.

Usefulness of Pidotimod and its role as immunostimulant, has been discussed, we know, for several decades. Nevertheless, there is still much to know. Understanding its mechanisms and its potential usefulness in airway infections and its prevention, asthma both Th2 and non Th2 type, bronchiectasis, as adjuvant in vaccination and in allergen immunotherapy still remains to clearly unveil. The aim of this paper was to provide a useful updated review of the role of the main available immunostimulants, giving particular focus on Pidotimod use and its potentials utility in respiratory diseases. Pidotimod showed its usefulness in reducing need for antibiotics in airway infections, increasing the level of immunoglobulins (IgA, IgM, IgG) and T-lymphocyte subsets (CD3+, CD4+) endowed with immunomodulatory activity that affect both innate and adaptive immune responses. Higher expression of TLR2 and of HLA-DR molecules, induction of dendritic cell maturation and release of pro-inflammatory molecules, stimulation of T lymphocyte proliferation and differentiation toward a Th1 phenotype, as well as an increase of the phagocytosis have been demonstrated to be associated with Pidotimod in in vitro studies. All these activities are potentially useful for several respiratory conditions such as asthma, COPD, and recurrent respiratory tract infections.

Asthma from immune pathogenesis to precision medicine.

Asthma is characterized by multiple immunological mechanisms (endotypes) determining variable clinical presentations (phenotypes). The identification of endotypic mechanisms is crucial to better characterize patients and to identify tailored therapeutic approaches with novel biological agents targeting specific immunological pathways. This review focused on summarizing the major immunological mechanisms involved in the pathogenesis of asthma, as well as on discussing the emergence of phenotypic features of the disease. Novel biological agents and other drugs targeting specific endotypes are discussed, as their use represent a precision medicine approach to the disease that is nowadays mandatory particularly for treating more severe patients.

Real-life studies of biologics used in asthma patients: key differences and similarities to trials.

Introduction: The precision medicine approach that is now mandatory for severe asthma management includes the use of novel biologic agents blocking specific immunological mechanisms that are responsible for disease phenotypes and endotypes: monoclonal antibodies blocking IgE, IL-5 and IL-4/IL-13 immunological pathways are so far available. Areas covered: Clinical trials involving a large number of patients proved their efficacy in reducing asthma exacerbations, improving lung function and quality of life, and reducing the need for systemic corticosteroid treatment. Since biologics have been available for routine use, a series of real-life experiences on severe asthmatics treated with them have been published: these studies confirmed the beneficial effects in a real-world setting (effectiveness) of these drugs and showed novel aspects that were not covered by clinical trials, such as their effect on particular subgroup of patients, unexpected adverse events, and potential novel indications. Expert opinion: Both clinical trials and real-life experiences are needed to establish robust data on biologic agents for severe asthma, with real-life studies giving more broader insights on different aspects related to the biologics themselves and to the disease.

Allergy and Sexual Behaviours: an Update.

The exact prevalence of hypersensitivity reactions related to sexual behaviours is not known; however, they heavily impact on the quality of life and of sex life of affected patients. In fact, not only common respiratory and skin allergies, such as asthma, rhinitis, urticaria and atopic dermatitis, but also food and drug allergy have been found to negatively affect the quality of sex life. Allergic diseases impact on the sexual function in both physical and psychological ways, representing one of the main complaints of a considerable proportion of patients. Sexual behaviours may act as the triggers of allergic reactions or as the carriers of allergens. Food and drug allergens can be carried through human organic fluids, like saliva and semen. Latex in condoms and numerous substances in lubricants, spermicides, topical medications and cosmetics can cause allergic reactions or contact dermatitis. Sexual activity itself is also a potential trigger of symptoms in patients affected by respiratory allergies, like honeymoon asthma and rhinitis. In seminal plasma hypersensitivity, seminal fluid proteins are the culprit allergens. The present review aims at summarizing the state of the art about allergy and sexual behaviours. In clinical practice, the influence of common allergic diseases on the sexual quality of life should be taken carefully into account. Sexual behaviours need to be accounted in the differential diagnosis of hypersensitivity reactions, and awareness on those exposure routes should be raised between different specialists and general practitioners.

Beer anaphylaxis due to coriander as hidden allergen.

Beer is one of the most widely consumed alcoholic beverage all over the world. Although the production and consumption of this beverage is diffuse, allergic reactions are very uncommonly reported, mainly due to wheat, yeast or hops allergy. More recently, many foods and drinks have been flavoured with spices, with a reported increase in allergic reactions. We report on a case of a young woman who experienced anaphylaxis due to coriander-flavoured beer. This is the first case of beer anaphylaxis due to coriander, which had been added to the beer as aromatising substance. As the presence of flavours is not always reported, they may be considered hidden allergens, whenever they are the cause of anaphylaxis to foods or beverages, the patient usually tolerated. In conclusion, allergic reactions to spices have to be considered in the patients with 'idiopathic' anaphylaxis induced by common foods, where spices had been hidden.

Basophil Membrane Expression of Epithelial Cytokine Receptors in Patients with Severe Asthma.

BACKGROUND: Severe asthma is a heterogeneous disease, which is characterized by airway damage and remodeling. All triggers of asthma, such as allergens, bacteria, viruses, and pollutants, interact with the airway epithelial cells, which drive the airway inflammatory response through the release of cytokines, particularly IL-25, IL-33, and thymic stromal lymphopoietin (TSLP). OBJECTIVES AND METHODS: To investigate whether the expression of the IL-25, IL-33, and TSLP receptors on the basophil membrane are associated with asthma severity. Twenty-six patients with asthma (11 severe and 15 moderate/mild) and 10 healthy subjects (controls) were enrolled in the study. The results of the basophil activation test and flow cytometry analysis were assessed to investigate basophil membrane expression of IL-25, TSLP, and IL-33 receptors before and after IgE stimulation. RESULTS: IL-25 and IL-33 receptor expression on the basophil membrane at baseline were significantly higher in patients with severe asthma than in those with mild/moderate asthma or healthy subjects, independent of atopy, eosinophilia, asthma control, and exacerbation frequency. Following IgE stimulation, a significantly higher increase in the IL-25 and IL-33 receptors was observed in mild/moderate versus severe asthma. CONCLUSIONS: The high expression of the IL-25 and IL-33 receptors on the basophil membrane of patients with severe asthma indicates an overstimulation of basophils by these cytokines in severe asthma. This finding can possibly be used as a biomarker of asthma severity.

Chronic Urticaria Patient Perspective (CUPP): The First Validated Tool for Assessing Quality of Life in Clinical Practice.

BACKGROUND: There is a need for validated tools to assess health-related quality of life (HRQoL) in routine clinical practice. OBJECTIVE: The aim of this study was to validate the Chronic Urticaria Patient Perspective (CUPP) for assessment of patients with chronic urticaria (CU) in clinical practice. METHODS: A provisional CUPP was developed from candidate items identified by following an iterative process in a retrospective analysis of 249 Chronic Urticaria Quality of Life Questionnaire questionnaires. The psychometric properties of the CUPP were then tested on a sample of patients enrolled in 13 Italian centers. RESULTS: The study population in the validation phase comprised 152 patients. The 10-item version of the CUPP showed satisfactory internal consistency (Cronbach's alpha values of 0.76 at visit 1 and 0.90 at visit 2), good criteria, and discriminative and convergent validity. Reliability was assessed in 34 patients with no changes in health (Global Rating Scale = 0 at visit 2) and was satisfactory (CCC [concordance correlation coefficient] = 0.9). Changes in CUPP scores were significantly associated with changes in Urticaria Activity Score (UAS)-Hive count (r = 0.36, P < .001), UAS-Itch severity (r = 0.48, P < .001), and UAS-Total score (r = 0.342, P < .001), all of which indicated good responsiveness. The minimal important difference was 1.5. CONCLUSIONS: CUPP is a simple 10-question tool with good psychometric properties that provides a valid, reliable, and standardized measurement of HRQoL in patients with CU.

Prevalence of and factors associated with prolonged length of stay in older hospitalized medical patients.

AIM: To characterize elderly medical patients and identify factors associated with prolonged length of stay. METHODS: The present prospective observational study evaluated consecutive patients aged ≥65 years admitted in acute geriatric and medical wards. A comprehensive assessment including demographic, clinical, functional and cognitive variables was carried out. Delayed discharge was defined when patients were discharged later than the date they were deemed medically ready for discharge by physicians. The analysis was initially carried out on the total sample and subsequently according to whether hospital admission had been from home, or from intermediate or long-term facilities. RESULTS: Among 1568 patients (age 81.3 ± 7.3 years, 712 men), we observed a high prevalence of functional dependence, cognitive impairment, chronic immobilization and frailty (50%, 25%, 20% and 40%, respectively). Overall, delayed discharge occurred in 442 cases - resulting in 2637 days of prolonged hospital stay - and was independently associated with impairment in activities of daily living, frailty, high comorbidity and inappropriate admission. Among patients admitted from home (roughly 90% of the sample), delayed discharge occurred in 392 patients, and was independently associated with cognitive impairment, functional dependence, low severity of comorbidity and inappropriate admission (OR 3.39). Among patients admitted from intermediate or long-term facilities, lower cognitive impairment and greater severity of functional dependence were independently associated with prolonged stay. CONCLUSIONS: Poor health conditions and high prevalence of geriatric syndromes are extremely common among older medical inpatients. Delayed discharge was mainly observed in patients admitted from home, and associated with cognitive impairment (OR 1.12) and functional dependence (OR 1.49).