Lucia Paolini

Abatacept Reduces Levels of Switched Memory B Cells, Autoantibodies, and Immunoglobulins in Patients with Rheumatoid Arthritis

Objective. Abatacept (ABA) is a chimeric molecule, able to block the CD28-mediated costimulatory pathway. To evaluate the hypothesis that, through this mechanism of action, ABA may down-modulate the immune responses of B lymphocytes in rheumatoid arthritis (RA), we investigated the serum levels of immunoglobulins (Ig), free light chains (FLC), anticitrullinated protein antibodies (ACPA), and rheumatoid factor (RF), as well as the number of B lymphocytes differentiated into post-switch memory cells in patients treated with ABA. Methods. The serum levels of Ig, FLC, different ACPA, RF isotypes, and the B cell phenotype were longitudinally evaluated in 30 patients with RA treated with ABA. Results. At baseline, the proportion of total and post-switch memory B cells was lower in RA than in healthy individuals. After 6 months of ABA treatment we observed significant reductions of serum levels of IgG, IgA, and IgM, as well as FLC, with a normalization in many patients who had initially abnormal values. A significant reduction of the titers of IgG- and IgA-ACPA, as well as of IgM-, IgA-, and IgG-RF was also observed. A decrease of autoantibodies below the upper limits of normal values was found in 2 of 26 patients (8%) initially seropositive for IgG-ACPA, 1 of 14 (7%) for IgA-ACPA, 5 of 22 (23%) for IgM-RF, 7 of 22 (30%) for IgA-RF, and 5 of 16 (31%) for IgG-RF. After treatment, the proportion of circulating post-switch memory B cells was also further significantly decreased. Conclusion. ABA treatment in patients with RA can reduce signs of polyclonal B cell activation, inducing a trend toward normalization of serum levels of different classes of Ig and of FLC, decreasing titers of ACPA and RF, and percentages of post-switch memory B cells.

Residual matrix from different separation techniques impacts exosome biological activity

Exosomes are gaining a prominent role in research due to their intriguing biology and several therapeutic opportunities. However, their accurate purification from body fluids and detailed physicochemical characterization remain open issues. We isolated exosomes from serum of patients with Multiple Myeloma by four of the most popular purification methods and assessed the presence of residual contaminants in the preparations through an ad hoc combination of biochemical and biophysical techniques - including Western Blot, colloidal nanoplasmonics, atomic force microscopy (AFM) and scanning helium ion microscopy (HIM). The preparations obtained by iodixanol and sucrose gradients were highly pure. To the contrary, those achieved with limited processing (serial centrifugation or one step precipitation kit) resulted contaminated by a residual matrix, embedding the exosomes. The contaminated preparations showed lower ability to induce NfkB nuclear translocation in endothelial cells with respect to the pure ones, probably because the matrix prevents the interaction and fusion of the exosomes with the cell membrane. These findings suggest that exosome preparation purity must be carefully assessed since it may interfere with exosome biological activity. Contaminants can be reliably probed only by an integrated characterization approach aimed at both the molecular and the colloidal length scales.

Colorimetric Nanoplasmonic Assay To Determine Purity and Titrate Extracellular Vesicles

Extracellular Vesicles (EVs) - cell secreted vesicles that carry rich molecular information of the parental cell and constitute an important mode of intercellular communication - are becoming a primary topic in translational medicine. EVs (that comprise exosomes and microvesicles/microparticles) have a size ranging from 40 nm to 1 μm and share several physicochemical proprieties, including size, density, surface charge, and light interaction, with other nano-objects present in body fluids, such as single and aggregated proteins. This makes separation, titration, and characterization of EVs challenging and time-consuming. Here we present a cost-effective and fast colorimetric assay for probing by eye protein contaminants and determine the concentration of EV preparations, which exploits the synergy between colloidal gold nanoplasmonics, nanoparticle-protein corona, and nanoparticle-membrane interaction. The assay hits a limit of detection of protein contaminants of 5 ng/μL and has a dynamic range of EV concentration ranging from 35 fM to 35 pM, which matches the typical range of EV concentration in body fluids. This work provides the first example of the exploitation of the nanoparticle-protein corona in analytical chemistry.

C-src Enriched Serum Microvesicles Are Generated in Malignant Plasma Cell Dyscrasia

Plasma cell dyscrasias are immunosecretory disorders that can lead to hematological malignancies such as Multiple Myeloma (MM). MM accounts for 15% of all hematologic cancers, and those diagnosed with MM typically become severely ill and have a low life expectancy. Monoclonal immunoglobulin Free Light Chains (FLC) are present in the serum and urine of many patients with plasma cell diseases. The biological differences between monoclonal FLCs, produced under malignant or benign dyscrasias, has not yet been characterized. In the present study, we show that endothelial and heart muscle cell lines internalize kappa and lambda FLCs. After internalization, FLCs are rerouted in the extracellular space via microvesicles and exosomes that can be re-internalized in contiguous cells. Only FLCs secreted from malignant B Lymphocytes were carried in Hsp70, annexin V, and c-src positive vesicles. In both MM and AL Amyloidosis patients we observed an increase in microvesicle and exosome production. Isolated serum vesicles from MM, AL Amyloidosis and monoclonal gammopathy of undetermined significance (MGUS) patients contained FLCs. Furthermore MM and AL amyloidosis vesicles were strongly positive for Hsp70, annexin V, and c-src compared to MGUS and control patients. These are the first data implying that FLCs reroute via microvesicles in the blood stream, and also suggest a potential novel mechanism of c-src activation in plasma cell dyscrasia.

Polyclonal versus monoclonal immunoglobulin-free light chains quantification

Background The clinical usefulness of the serum-free light chain assays has expanded since their first description, and further applications other than plasma cell dyscrasia are emerging. Currently, we have the ability to perform the measurements with two certified methods: the Freelite™ assay (The Binding Site Ltd, Birmingham, UK) and the new N Latex free-light chain assay (Siemens, Germany). In the present study, we investigated the impact of free light chain concentrations and structures on their quantification, performed with both tests. Methods A total of 524 serum samples from 497 patients from our routine laboratory were analysed with the Freelite™ and the N Latex free light chain assay. The results were compared in two subgroups: with or without monoclonal component. Twenty-four samples were subsequently investigated for the presence of dimeric and monomeric free light chain with sodium dodecyl sulphate polyacrylamide gel electrophoresis and densitometric quantification. Results Methods comparison showed that the Pearson rank correlation coefficients were 0.90 for polyclonal k and 0.91 for polyclonal λ free light chain. Conversely for monoclonal immunoglobulins, the Pearson rank correlation coefficient was lower with 0.82 for kM >500 mg/L and 0.56 for λM >500 mg/L. Furthermore, densitometric quantification of the involved monoclonal free light chains showed that both assays do not reflect the Coomassie-stained protein mass. Conclusion Samples containing high amounts of a single pathologic free light chain may not be considered like a sample containing a sum of different polyclonal free light chains. Indeed, free light chain dimerization leads to different scatter efficiency of macromolecular complexes.

Immunoglobulin free light chains and GAGs mediate Multiple Myeloma Extracellular Vesicles uptake and secondary NfkB nuclear translocation

Multiple myeloma (MM) is a hematological malignancy caused by a microenviromentally aided persistence of plasma cells in the bone marrow. Monoclonal plasma cells often secrete high amounts of immunoglobulin free light chains (FLCs) that could induce tissue damage. Recently, we showed that FLCs are internalized in endothelial and myocardial cell lines and secreted in extracellular vesicles (EVs). MM serum derived EVs presented phenotypic differences if compared with monoclonal gammopathy of undetermined significance (MGUS) serum derived EVs suggesting their involvement in MM pathogenesis or progression. To investigate the effect of circulating EVs on endothelial and myocardial cells, we purified MM and MGUS serum derived EVs with differential ultracentrifugation protocols and tested their biological activity. We found that MM and MGUS EVs induced different proliferation and internalization rates in endothelial and myocardial cells, thus we tried to find specific targets in MM EVs docking and processing. Pre-treatment of EVs with anti-FLCs antibodies or heparin blocked the MM EVs uptake, highlighting that FLCs and glycosaminoglycans are involved. Indeed, only MM EVs exposure induced a strong nuclear factor kappa B nuclear translocation that was completely abolished after anti-FLCs antibodies and heparin pre-treatment. The protein tyrosine kinase c-src is present on MM circulating EVs and redistributes to the cell plasma membrane after MM EVs exposure. The anti-FLCs antibodies and heparin pre-treatments were able to block the intracellular re-distribution of the c-src kinase and the subsequent c-src kinase containing EVs production. Our results open new insights in EVs cellular biology and in MM therapeutic and diagnostic approaches.

The Epsilon Hinge-Ear Region Regulates Membrane Localization of the AP-4 Complex

Adaptor protein (AP) complexes are key factors for the spatial and temporal regulation of intracellular trafficking events. Four complexes (AP‐1, ‐2, ‐3, ‐4) are known, among which AP‐4 is only poorly characterized. Recent work suggests a role for AP‐4 in the intracellular trafficking of the β‐amyloid precursor protein and molecular genetics showed that the loss of functional AP‐4 is associated with congenital neuronal disorders of severe cognitive dysfunction. To unravel the molecular mechanisms controlling AP‐4 functions, we established the intracellular expression of recombinant AP‐4 complex. This approach combined with the analysis of mutant complexes allowed us to discover that the epsilon adaptin hinge‐ear region has a function in membrane recruitment of AP‐4. We further show that this process is phosphorylation dependent and involves PP2A‐like protein phosphatases and a staurosporine‐sensitive kinase. Deletion of the residues 839‐871 in the carboxy‐terminal region of the hinge of epsilon adaptin abrogated the membrane/cytosol recycling of AP‐4. As targets of phosphorylation, we identified three serine residues: S847, S868 and S871. We conclude that the terminal hinge region and the appendage of the AP‐4 epsilon subunit are involved in membrane association in a process that is controlled by phosphorylation and dephosphorylation events.

Comparison of Hevylite™ IgA and IgG assay with conventional techniques for the diagnosis and follow-up of plasma cell dyscrasia

Background Heavy/light chain assay allows the characterization and quantification of immunoglobulin light chains bound to heavy chains for each Ig’k and Ig’λ immunoglobulin class, discriminating between the involved/uninvolved isotypes in plasma cell dyscrasia. The Ig’k/Ig’λ ratio (heavy/light chain ratio) enables to monitor the trend of monoclonal component during therapy and disease evolution. Objective In this study, we evaluate the impact of the heavy/light chain assay in monitoring multiple myeloma patients in comparison with conventional techniques. Methods Serum samples of 28 patients with IgG or IgA monoclonal component were collected for a mean of 109 days and analyzed. The heavy/light chain assay was compared with classical immunoglobulin quantification (Ig’Tot), serum immunofixation electrophoresis, serum protein electrophoresis, and serum-free light chains quantification. Serum samples from 30 healthy patients were used as control (polyclonal). Results Heavy/light chain ratio and serum immunofixation electrophoresis were comparable in 86% of the cases, and free light chain ratio and heavy/light chain ratio in 71.8%. Heavy/light chain assay and Ig’Tot measurements showed a concentration-dependent agreement in monoclonal patients. The heavy/light chain assay was able to quantify the monoclonal component migrating in SPE β region: this occurred in 10% of our IgG and 50% of our IgA patients. Conclusions The concordance scores indicate that heavy/light chain and Ig’Tot assays show differences at high monoclonal component values. The heavy/light chain ratio, serum immunofixation electrophoresis, and free light chain ratio showed partial concordance. Our study confirmed that, in the context of heavy/light chain assay, heavy/light chain Ig’k and Ig’λ absolute values and heavy/light chain ratio are both important tools to monitor the presence of monoclonal component that are difficult to be identified in SPE.

Exosomes Secreted by HeLa Cells Shuttle on Their Surface the Plasma Membrane-Associated Sialidase NEU3

Sialidases are glycohydrolases that remove terminal sialic acid residues from oligosaccharides, glycolipids, and glycoproteins. The plasma membrane-associated sialidase NEU3 is involved in the fine-tuning of sialic acid-containing glycans directly on the cell surface and plays relevant roles in important biological phenomena such as cell differentiation, molecular recognition, and cancer transformation. Extracellular vesicles are membranous structures with a diameter of 0.03-1 μm released by cells and can be detected in blood, urine, and culture media. Among extracellular vesicles, exosomes play roles in intercellular communication and maintenance of several physiological and pathological conditions, including cancer, and could represent a useful diagnostic tool for personalized nanomedicine approaches. Using inducible expression of the murine form of NEU3 in HeLa cells, a study of the association of the enzyme with exosomes released in the culture media has been performed. Briefly, NEU3 is associated with highly purified exosomes and localizes on the external leaflet of these nanovesicles, as demonstrated by enzyme activity measurements, Western blot analysis, and dot blot analysis using specific protein markers. On the basis of these results, it is plausible that NEU3 activity on exosome glycans enhances the dynamic biological behavior of these small extracellular vesicles by modifying the negative charge and steric hindrance of their glycocalyx. The presence of NEU3 on the exosomal surface could represent a useful marker for the detection of these nanovesicles and a tool for improving our understanding of the biology of these important extracellular carriers in physiological and pathological conditions.

Size distribution of extracellular vesicles by optical correlation techniques

Understanding the colloidal properties of extracellular vesicles (EVs) is key to advance fundamental knowledge in this field and to develop effective EV-based diagnostics, therapeutics and devices. Determination of size distribution and of colloidal stability of purified EVs resuspended in buffered media is a complex and challenging issue - because of the wide range of EV diameters (from 30 to 2000nm), concentrations of interest and membrane properties, and the possible presence of co-isolated contaminants with similar size and densities, such as protein aggregates and fat globules - which is still waiting to be fully addressed. We report here a fully detailed protocol for accurate and robust determination of the size distribution and stability of EV samples which leverages a dedicated combination of Fluorescence Correlation Spectroscopy (FCS) and Dynamic Light Scattering (DLS). The theoretical background, critical experimental steps and data analysis procedures are thoroughly presented and finally illustrated through the representative case study of EV formulations obtained from culture media of B16 melanoma cells, a murine tumor cell line used as a model for human skin cancers.