Considering the important advances in treating specific types of systemic amyloidoses, unequivocal typing of amyloid deposits is now essential. Subcutaneous abdominal fat aspiration is the easiest, most common diagnostic procedure. We developed a novel, automated approach, based on Multidimensional Protein Identification Technology, for typing amyloidosis. Fat aspirates were obtained from patients with the most common systemic amyloidoses (ALλ, ALκ, transthyretin, and reactive amyloidosis), with Congo red score more than or equal to 3+, and nonaffected controls. Peptides from extracted and digested proteins were analyzed by Multidimensional Protein Identification Technology. On semiquantitative differential analysis (patients vs controls) of mass spectrometry data, specific proteins up-represented in patients were identified and used as deposit biomarkers. An algorithm was developed to classify patients according to type and abundance of amyloidogenic proteins in samples; in all cases, proteomic characterization was concordant with fibril identification by immunoelectron microscopy and consistent with clinical presentation. Our approach allows reliable amyloid classification using readily available fat aspirates.

In this paper, a pilot production process for mesenchymal stem/stromal freeze-dried secretome was performed in a validated good manufacturing practice (GMP)-compliant cell factory. Secretome was purified from culture supernatants by ultrafiltration, added to cryoprotectant, lyophilized and characterized. We obtained a freeze-dried, “ready-off-the-shelf” and free soluble powder containing extracellular vesicles and proteins. In the freeze-dried product, a not-aggregated population of extracellular vesicles was detected by nanoparticle tracking analysis; Fourier transform infrared spectra showed the simultaneous presence of protein and lipids, while differential scanning calorimetry demonstrated that lyophilization process successfully occurred. A proteomic characterization allowed the identification of proteins involved in immune response, response to stress, cytoskeleton and metabolism. Moreover, the product was not cytotoxic up to concentrations of 25 mg/mL (on human fibroblasts, chondrocytes and nucleus pulposus cells by MTT assay) and was blood compatible up to 150 mg/mL. Finally, at concentrations between 5 and 50 mg/mL, freeze-dried secretome showed to in vitro counteract the oxidative stress damage induced by H₂O₂ on nucleus pulposus cells by MTT assay.

Asthma is a chronic inflammatory disease. Reticular basement membrane (RBM) thickening is considered feature of airway remodelling (AR) particularly in severe asthma (SA). Omalizumab, mAb to IgE is effective in SA and can modulate AR. Herein we describe protein profiles of bronchial biopsies to detect biomarkers of anti-IgE effects on AR and to explain potential mechanisms/pathways. We defined the bronchial biopsy protein profiles, before and after treatment. Unsupervised clustering of baseline proteomes resulted in very good agreement with the morphometric analysis of AR. Protein profiles of omalizumab responders (ORs) were significantly different from those of non-omalizumab responders (NORs). The major differences between ORs and NORs lied to smooth muscle and extra cellular matrix proteins. Notably, an IgE-binding protein (galectin-3) was reliable, stable and predictive biomarker of AR modulation. Omalizumab down-regulated bronchial smooth muscle proteins in SA. These findings suggest that omalizumab may exert disease-modifying effects on remodelling components.

Bronchial asthma is a heterogeneous disease characterized by three cardinal features: chronic inflammation, variable airflow obstruction, and airway hyperresponsiveness. Asthma has traditionally been defined using nonspecific clinical and physiologic variables that encompass multiple phenotypes and are treated with nonspecific anti-inflammatory therapies. Based on the modulation of airway remodeling after 12 months of anti-immunoglobulin E (IgE) treatment, we identified two phenotypes (omalizumab responder, OR; and non-omalizumab responder, NOR) and performed morphometric analysis of bronchial biopsy specimens. We also found that these two phenotypes were correlated with the presence/absence of galectin-3 (Gal-3) at baseline (i.e., before treatment). The aims of the present study were to investigate the histological and molecular effects of long-term treatment (36 months) with anti-IgE and to analyze the behavior of OR and NOR patients. All patients were treated with the monoclonal antibody anti-IgE omalizumab for 36 months. The bronchial biopsy specimens were evaluated using morphometric, eosinophilic, and proteomic analysis (MudPIT). New data were compared with previous data, and unsupervised cluster analysis of protein profiles was performed. After 36 months of treatment with omalizumab, reduction of reticular basement membrane (RBM) thickness was confirmed in OR patients (Gal-3-positive at baseline); similarly, the protein profiles (over 500 proteins identified) revealed that, in the OR group, levels of proteins specifically related to fibrosis and inflammation (e.g., smooth muscle and extracellular matrix proteins (including periostin), Gal-3, and keratins decreased by between 5- and 50-fold. Eosinophil levels were consistent with molecular data and decreased by about tenfold less in ORs and increased by twofold to tenfold more in NORs. This tendency was confirmed (p < 0.05) based on both fold change and DAVE algorithms, thus indicating a clear response to anti-IgE treatment in Gal-3-positive patients. Our results showed that omalizumab can be considered a disease-modifying treatment in OR. The proteomic signatures confirmed the presence of Gal-3 at baseline to be a biomarker of long-term reduction in bronchial RBM thickness, eosinophilic inflammation, and muscular and fibrotic components in omalizumab-treated patients with severe asthma. Our findings suggest a possible relationship between Gal-3 positivity and improved pulmonary function.

Accumulating evidence shows that Mesenchymal Stem/Stromal Cells (MSCs) exert their therapeutic effects by the release of secretome, made of both soluble proteins and nano/microstructured extracellular vesicles (EVs). In this work, for the first time, we proved by a proteomic investigation that adipose-derived (AD)-MSC-secretome contains alpha-1-antitrypsin (AAT), the main elastase inhibitor in the lung, 72 other proteins involved in protease/antiprotease balance, and 46 proteins involved in the response to bacteria. By secretome fractionation, we proved that AAT is present both in the soluble fraction of secretome and aggregated and/or adsorbed on the surface of EVs, that can act as natural carriers promoting AAT in vivo stability and activity. To modulate secretome composition, AD-MSCs were cultured in different stimulating conditions, such as serum starvation or chemicals (IL-1β and/or dexamethasone) and the expression of the gene encoding for AAT was increased. By testing in vitro the anti-elastase activity of MSC-secretome, a dose-dependent effect was observed; chemical stimulation of AD-MSCs did not increase their secretome anti-elastase activity. Finally, MSC-secretome showed anti-bacterial activity on Gram-negative bacteria, especially for Klebsiella pneumoniae. These preliminary results, in addition to the already demonstrated immunomodulation, pave the way for the use of MSC-secretome in the treatment of AAT-deficiency lung diseases.

Boron neutron capture therapy (BNCT) is a binary cancer treatment modality where two different agents (¹⁰B and thermal neutrons) have to be present to produce an effect. A dedicated trial design is necessary for early clinical trials. The concentration of ¹⁰B in tissues is an accepted surrogate to predict BNCT effects on tissues. Tissue, blood, and urines were sampled after infusion of two different boron carriers, namely BSH and BPA in the frame of the European Organisation for Research and Treatment of Cancer (EORTC) trial 11001. In this study, urine samples were used to identify protein profiles prior and after drug infusion during surgery. Here, an approach that is based on the mass spectrometry (MS)-based proteomic analysis of urine samples from head and neck squamous cell carcinoma (HNSCC) and thyroid cancer patients is presented. This method allowed the identification of several inflammation- and cancer-related proteins, which could serve as tumor biomarkers. In addition, changes in the urinary proteome during and after therapeutic interventions were detected. In particular, a reduction of three proteins that were involved in inflammation has been observed: Galectin-3 Binding Protein, CD44, and osteopontin. The present work represents a proof of principle to follow proteasome changes during complex treatments based on urine samples.