Abstract Helminths survive within their host by secreting immunomodulatory compounds, which hold therapeutic potential for inflammatory conditions. Helminth‐derived extracellular vesicles (EVs) are one such component proposed to possess immunomodulatory activities. Due to the recent discovery of helminth EVs, standardised protocols for EV separation are lacking. Excretory/secretory products of the porcine helminth, Ascaris suum , were used to compare three EV separation methods: Size exclusion chromatography (SEC), ultracentrifugation (UC) and a combination of the two. Their performance was evaluated by EV yield, sample purity and the ability of EVs to suppress lipopolysaccharide (LPS)‐induced inflammation in vitro. We found that all three separation methods successfully separated helminth EVs with a similar EV yield. Functional studies showed that EVs from all three methods reduced LPS‐induced levels of tumour necrosis factor (TNF‐α) in a dose‐dependent manner. Overall, the three separation methods showed similar performance, however, the combination of UC+SEC presented with slightly higher purity than either method alone.
Abstract Parasitic helminths secrete extracellular vesicles (EVs) into their host tissues to modulate immune responses, but the underlying mechanisms are poorly understood. We demonstrate that Ascaris EVs are efficiently internalised by monocytes in human peripheral blood mononuclear cells and increase the percentage of classical monocytes. Furthermore, EV treatment of monocytes induced a novel anti‐inflammatory phenotype characterised by CD14 + , CD16 − , CC chemokine receptor 2 (CCR2 − ) and programmed death‐ligand 1 (PD‐L1) + cells. In addition, Ascaris EVs induced T cell anergy in a monocyte‐dependent mechanism. Targeting professional phagocytes to induce both direct and indirect pathways of immune modulation presents a highly novel and efficient mechanism of EV‐mediated host‐parasite communication. Intra‐peritoneal administration of EVs induced protection against gut inflammation in the dextran sodium sulphate model of colitis in mice. Ascaris EVs were shown to affect circulating immune cells and protect against gut inflammation; this highlights their potential as a subject for further investigation in inflammatory conditions driven by dysregulated immune responses. However, their clinical translation would require further studies and careful consideration of ethical implications.
The Biomolecular Corona (BC) is a typical phenomenon that occurs at the interface between the nanomaterial and the surrounding media, involving the adsorption or the desorption of macromolecules at this interface. Studying BC dynamic properties on soft nanomaterials (i.e., liposomes or extracellular vesicles) proves to be challenging. Advancing in situ methodologies for investigating BC on soft nanoparticles is essential for elucidating the intricate interplay between surface composition, properties, and dynamic BC formation. In this study, we present new evidence of distinct dynamics in BC formation for two extracellular vesicle (EV) subtypes. Utilizing Fluorescence Correlation Spectroscopy (FCS) coupled with a fluorescent monoclonal antibody as a corona probe enabled the characterization of macromolecule exchange at the EV surface and the dynamic evolution of the BC.
Serum protein electrophoresis (SPE), serum immunofixation (s-IFE), free light chain measurement (FLC) and nephelometric measurements of total immunoglobulin in serum (IgTot) are some of the laboratory tests required for the management of plasma cell proliferative disorders. The monoclonal protein is usually visible on SPE as a spike (M-spike) in the γ region and the derived densitogram is used to quantify it relative to serum total protein concentration. IgA M-protein, however, often migrates in the β region on SPE and its quantification can be masked by other serum proteins that migrate in this region. The immunoassay Hevylite™ (heavy/light chain, HLC) seems to solve this problem: it quantifies the involved/uninvolved isotype, calculating the ratio IgAκ/IgAλ, considered indicative of clonal proliferation. However, this test seems redundant in the case of artifacts on SPE such as obvious hemolysis or lipemia, or if the IgA M-spike is clearly visible in the β region. In conclusion whereas the IgA HLC assay does not represent an alternative to SPE and s-IFE in the diagnostic patient workup, it may prove to be an alternative to SPE, s-IFE and total IgA quantification in risk stratification and evaluation of response to therapy in patients affected by MM and other monoclonal plasma proliferative disorders.
Extracellular vesicles (EVs) are cell-derived nanoparticles, involved in cell-to-cell communication, in both normal and pathological processes. Originating by the outward budding of the plasma membrane or released by exocytosis, they are natural cargoes for lipids, carbohydrates, proteins and nucleic acids. EV-based diagnostics promises unique advantages compared with conventional strategies involving whole body fluid analysis, including the reduction of biofluids complexity and more specific and sensitive detection of low abundance biomacromolecules. Besides EV cargoes, new breakthrough technologies are addressing EV 'colloidal properties' – including particle content, size and membrane mechanical properties – directly experienced by researchers to be critical factors in biomarkers discovery. This article focuses on the progresses in EV biophysical properties characterization as diagnostic tools for different pathological conditions.
Through costimulation blockade, Abatacept (ABA) may down-modulate the immune responses of B lymphocytes in Rheumatoid Arthritis (RA). We previously showed that ABA reduces the serum levels of different classes of anti-cyclic citrullinated peptide (anti-CCP), and rheumatoid factor (RF), and limits the differentiation of B lymphocytes into post-switch memory cells (1). Free light chains (FLC) are produced by B cells, plasmablasts and plasmacells and raised levels correlate with disease activity in RA.
Objectives
To evaluate the effect of the ABA on serum levels of immunoglobulins (Ig) and FLC. To evaluate the baseline levels of these parameters as predictors of response to ABA.
Methods
30 RA patients (pts) (26 female; median-age: 53 years [IQR: 44-60] treated for at least 6 months with ABA and 24 healthy controls (HC; 18 female; age: 39 [34-46]) were evaluated. Serum Ig levels were measured by a nephelometric-immunoassay (Siemens Healthcare Diagnostics Products GmbH). Reference ranges were derived by a consensus group based on the standardization against the calibrated reference material CRM 470. Serum FLC levels were measured by a latex-enhanced immunoassay (The Binding Site, Birmingham, UK). Reference ranges include 100% of a population of 282 HC. Clinical disease activity was evaluated with the DAS28 (based on CRP).
Results
At baseline (T0), RA pts had higher serum levels of IgM (p<0.05), IgA (p<0.001), free k chains (p<0.001), free λ chains (p<0.01), and k:λ ratio (p<0.01) than HC. In comparison with reference ranges, raised levels of serum IgG, IgA, IgM were observed in 17%, 37%, and 20% of RA pts, respectively; no one had hypogammaglobulinemia. Raised levels of k and λ FLC were demonstrated in 68% and 17% of pts. The k:λ ratio was above normal levels in 27% of them. After 6 months of ABA (T6), 53% of patients achieved the clinical remission; also a significant reduction of serum levels of IgG (p<0.001), IgA (p<0.001), and IgM (p<0.01) was seen. Baseline abnormal values of IgG, IgA, and IgM normalized at T6 in 2 of 5, 3 of 11 and 4 of 6 pts, respectively. Analogously, serum free k and λ chains decreased significantly at T6 (p<0.01 and p:0.01), normalizing respectively in 6 of 18, and 4 of 5 patients with raised levels at T0. The k:λ ratio was also significantly reduced after therapy (p:0.02), normalizing in 4 of 8 patients with raised ratio at T0. The decrease of FLC was significant only in patients with clinical remission at T6, but not in those without, whereas serum IgG and IgA levels decreased in both groups of patients. Moreover, the reduction of free λ chains was significantly correlated with the reduction of DAS28-CRP (r:0.47; p:0.01). Evaluating predicting biomarkers of response, baseline free λ chains serum levels were lower in patients achieving clinical remission at T6 than in the other pts (p:0.045).
Conclusions
ABA therapy induced a trend toward normalization of serum levels of total Ig of different classes and of FLC. FLC reduction was significant in pts achieving clinical remission after ABA therapy, but not in those who did not. In our evaluation, the decrease of serum free λ chains was correlated with clinical improvement, and their baseline levels appeared to be associated with clinical response to ABA.
To be profitably exploited in medicine as drug delivery carriers, nanosized systems should be endowed with essential properties, including high biocompatibility, stealth ability to escape the immune system, prolonged circulation lifetime, and targeting abilities towards the pathological tissues. Currently, the choice of biogenic nanoparticles as Extracellular Vesicles (EVs) represents the most promising strategy to deal with clearance issues. EV targeting properties and pharmacokinetics could be augmented by surface engineering methods by inserting specific target ligands, including antibodies or small peptides. Biorthogonal chemistry allows for the formation of a stable covalent bond among these moieties and EV surface, ensuring the maintenance of biomolecule’s reactivity. As for synthetic nanoparticle counterparts, it has recently been acknowledged proteins might physically adsorb on the EV surface in biofluids, forming a protein corona (PC), thus modifying EV biological identity. This phenomenon cannot be overlooked when investigating the properties and behavior of nanosized systems in biological media. In this work, we explored the possibility to engineer the EV surface with a modified antibody (Cetuximab, CTX) by both chemisorption (via click chemistry reaction) and physisorption (forming a protein corona) mechanisms. We found that both mechanisms are suitable to engineer the EV surface and, at the molecular level, SPR analysis showed that EV formulations functionalized by CTX, through its physisorption or chemisorption presented a comparable affinity for EGFR. However, at the cellular level, the improved stability for CTX-EV prepared by click chemistry strategies confers superior binding and uptake ability toward target cells. These results highlight for the first time the EV-PC formation during surface engineering processes and suggest its functional role, which should be carefully estimated in EV-based drug delivery system development due to the lower stability of the EV-PC compared to the covalent chemical binding between EV and CTX.
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.
<div>Abstract<p>Melanoma is an immunogenic neoplasm infiltrated by T cells, although these adaptive T cells usually fail to eradicate the tumor. Plasmacytoid dendritic cells (PDCs) are potent regulators of the adaptive immune response and can eliminate melanoma cells via TLR-mediated effector functions. The PDC compartment is maintained by progressively restricted bone marrow progenitors. Terminally differentiated PDCs exit the bone marrow into the circulation, then home to lymph nodes and inflamed peripheral tissues. Infiltration by PDCs is documented in various cancers. However, their role within the melanoma immune contexture is not completely known. We found that in locoregional primary cutaneous melanoma (PCM), PDC infiltration was heterogeneous, occurred early, and was recurrently localized at the invasive margin, the site where PDCs interact with CD8<sup>+</sup> T cells. A reduced PDC density was coupled with an increased Breslow thickness and somatic mutations at the <i>NRAS</i> p.Q61 codon. Compared with what was seen in PCM, high numbers of PDCs were found in regional lymph nodes, as also identified by <i>in silico</i> analysis. In contrast, in metastatic melanoma patients, PDCs were mostly absent in the tumor tissues and were significantly reduced in the circulation, particularly in the advanced M1c group. Exposure of circulating PDCs to melanoma cell supernatant (SN-mel) depleted of extracellular vesicles resulted in significant PDC death. SN-mel exposure also resulted in a defect of PDC differentiation from CD34<sup>+</sup> progenitors. These findings indicate that soluble components released by melanoma cells support the collapse of the PDC compartment, with clinical implications for refining TLR agonist–based trials.</p></div>
