Tom Groot Kormelink

EV-TRACK: transparent reporting and centralizing knowledge in extracellular vesicle research

We argue that the field of extracellular vesicle (EV) biology needs more transparent reporting to facilitate interpretation and replication of experiments. To achieve this, we describe EV-TRACK, a crowdsourcing knowledgebase (http://evtrack.org) that centralizes EV biology and methodology with the goal of stimulating authors, reviewers, editors and funders to put experimental guidelines into practice.

Antigen-specific, antibody-coated, exosome-like nanovesicles deliver suppressor T-cell microRNA-150 to effector T cells to inhibit contact sensitivity

BACKGROUND T-cell tolerance of allergic cutaneous contact sensitivity (CS) induced in mice by high doses of reactive hapten is mediated by suppressor cells that release antigen-specific suppressive nanovesicles. OBJECTIVE We sought to determine the mechanism or mechanisms of immune suppression mediated by the nanovesicles. METHODS T-cell tolerance was induced by means of intravenous injection of hapten conjugated to self-antigens of syngeneic erythrocytes and subsequent contact immunization with the same hapten. Lymph node and spleen cells from tolerized or control donors were harvested and cultured to produce a supernatant containing suppressive nanovesicles that were isolated from the tolerized mice for testing in active and adoptive cell-transfer models of CS. RESULTS Tolerance was shown due to exosome-like nanovesicles in the supernatants of CD8(+) suppressor T cells that were not regulatory T cells. Antigen specificity of the suppressive nanovesicles was conferred by a surface coat of antibody light chains or possibly whole antibody, allowing targeted delivery of selected inhibitory microRNA (miRNA)-150 to CS effector T cells. Nanovesicles also inhibited CS in actively sensitized mice after systemic injection at the peak of the responses. The role of antibody and miRNA-150 was established by tolerizing either panimmunoglobulin-deficient JH(-/-) or miRNA-150(-/-) mice that produced nonsuppressive nanovesicles. These nanovesicles could be made suppressive by adding antigen-specific antibody light chains or miRNA-150, respectively. CONCLUSIONS This is the first example of T-cell regulation through systemic transit of exosome-like nanovesicles delivering a chosen inhibitory miRNA to target effector T cells in an antigen-specific manner by a surface coating of antibody light chains.

Evidence for the involvement of free light chain immunoglobulins in allergic and nonallergic rhinitis

BACKGROUND Allergic rhinitis is characterized by mast cell degranulation induced by antigen cross-linking of IgE. It has been proposed that some patients with rhinitis show nasal allergy in the absence of systemic markers of atopy, termed entopy. Recent murine studies suggest the existence of an IgE-independent hypersensitivity response involving antigen-induced mast cell activation, mediated by immunoglobulin free light chains (FLCs). OBJECTIVES To determine whether FLC is associated with mast cell-mediated nasal hypersensitivity and its relationship with eosinophilic activity in allergic and nonatopic rhinitis. METHODS Patients with allergy and nonallergic rhinitis with eosinophilia syndrome (NARES) had levels of soluble FLC measured in nasal secretions and serum. In addition, levels of the nasal inflammatory mediators mast cell tryptase and eosinophil cationic protein were quantified. Cellular expression of kappa and lambda FLC was characterized in the nasal mucosa of allergic and nonatopic idiopathic rhinitis and control subjects by using immunohistochemistry. Immunopositive cells were phenotyped by using laser microdissection and PCR. RESULTS Free light chain was significantly increased in nasal secretions of subjects with allergy and NARES, and in serum of patients with NARES. Nonatopic patients with allergy showed significantly increased nasal mast cell tryptase and eosinophil cationic protein. FLC-positive cells were significantly increased in allergic and nonatopic mucosa, and were shown to be mast cells and plasma cells. CONCLUSION Nasal FLC is significantly increased in allergic and nonatopic rhinitis nasal mucosa, suggesting a role in nasal hypersensitivity. Further studies are needed to identify which allergens trigger FLC-mediated responses in nonatopic rhinitis.

Mast Cells as Target in Cancer Therapy

A close interaction of cancer cells with their microenvironment is important for their growth and survival. In this respect, the involvement of inflammatory cells in the initiation, promotion and progression of cancer has pointed to new therapeutic opportunities in the treatment of cancer. The main immune cell types implicated in tumor-associated inflammation are macrophages, dendritic cells, lymphocytes, neutrophils, eosinophils and mast cells. Their precise role in intercellular communication, regulation of tumor inflammation, and to what respect this inflammation contributes to tumor development, are not completely understood. Mast cells are key effector cells in allergic diseases, but it has become apparent that they also contribute to other pathologies, including autoimmune diseases and cancer. Activated mast cells can release many pro-angiogenic and tumor growth stimulatory mediators. Increased numbers of mast cells are found in many tumors and it has been shown that the number of tumor infiltrating mast cells correlate with increased intratumoral microvessel density, enhanced tumor growth and tumor invasion, and poor clinical outcome. Therefore, modulating mast cell recruitment, viability, activity, or mediator release patterns at malignant sites can be of importance to control tumor growth. In this review, we will focus on the contribution of mast cells to tumor development and growth and the possibilities to interfere in mast cell activation and proliferation in the therapy of cancer.

Contribution of IgE and immunoglobulin free light chain in the allergic reaction to cow's milk proteins

BACKGROUND Cows milk allergy (CMA) affects 2.5% of young infants. In previous murine studies it was observed that allergic sensitization to the major cows milk allergens casein and whey led, respectively, to IgE-independent and IgE-dependent clinical responses. OBJECTIVES In this study the involvement of immunoglobulin free light chains (Ig-fLCs) in the hypersensitivity response to cows milk proteins was explored in mice, and Ig-fLC serum levels were determined in children affected by CMA or atopic dermatitis (AD). METHODS Mice were orally sham, casein, or whey sensitized. Acute allergen-specific skin responses were determined, and serum immunoglobulin and Ig-fLC concentrations were measured. Ig-fLC dependency was validated by using the Ig-fLC blocker F991 in actively and passively sensitized mice. Ig-fLC serum concentrations were measured in a cohort of infants with CMA and infants with AD. RESULTS After sensitization, no specific IgE was detectable in sera of casein-sensitized mice, whereas specific IgE levels were enhanced in whey-sensitized mice. Instead, Ig-fLC levels were increased in sera from casein-sensitized mice. Furthermore, blocking Ig-fLCs strongly diminished the allergic skin responses not only in casein-sensitized mice but also in mice transferred with splenocyte supernatants of casein-sensitized mice. In both patients with CMA and patients with AD, serum Ig-fLC concentrations were significantly enhanced. CONCLUSIONS This study indicates that sensitization with cows milk proteins can lead to both IgE-dependent and Ig-fLC-dependent allergic hypersensitivity responses. Also, in children affected with CMA or AD, serum Ig-fLC concentrations were increased, implying the relevance of Ig-fLC measurements in the diagnoses of human allergic disease.

Prerequisites for the analysis and sorting of extracellular vesicle subpopulations by high-resolution flow cytometry

Submicron‐sized vesicles released by cells are increasingly recognized for their role in intercellular communication and as biomarkers of disease. Methods for high‐throughput, multi‐parameter analysis of such extracellular vesicles (EVs) are crucial to further investigate their diversity and function. We recently developed a high‐resolution flow cytometry‐based method (using a modified BD Influx) for quantitative and qualitative analysis of EVs. The fact that the majority of EVs is <200 nm in size requires special attention with relation to specific conditions of the flow cytometer, as well as sample concentration and event rate. In this study, we investigated how (too) high particle concentrations affect high‐resolution flow cytometry‐based particle quantification and characterization. Increasing concentrations of submicron‐sized particles (beads, liposomes, and EVs) were measured to identify coincidence and swarm effects, caused by the concurrent presence of multiple particles in the measuring spot. As a result, we demonstrate that analysis of highly concentrated samples resulted in an underestimation of the number of particles and an interdependent overestimation of light scattering and fluorescence signals. On the basis of this knowledge, and by varying nozzle size and sheath pressure, we developed a strategy for high‐resolution flow cytometric sorting of submicron‐sized particles. Using the adapted sort settings, subsets of EVs differentially labeled with two fluorescent antibodies could be sorted to high purity. Moreover, sufficient numbers of EVs could be sorted for subsequent analysis by western blotting. In conclusion, swarm effects that occur when measuring high particle concentrations severely hamper EV quantification and characterization. These effects can be easily overlooked without including proper controls (e.g., sample dilution series) or tools (e.g., oscilloscope). Providing that the event rate is well controlled, the sorting strategy we propose here indicates that high‐resolution flow cytometric sorting of different EV subsets is feasible.

Non-digestible oligosaccharides reduce immunoglobulin free light-chain concentrations in infants at risk for allergy.

To cite this article: Schouten B, Van Esch BCAM, Kormelink TG, Moro GE, Arslanoglu S, Boehm G, Knippels LMJ, Redegeld FA, Willemsen LEM, Garssen J. Non‐digestible oligosaccharides reduce immunoglobulin free light‐chain concentrations in infants at risk for allergy. Pediatr Allergy Immunol 2011; 22: 537–542.

An Association between Neutrophils and Immunoglobulin Free Light Chains in the Pathogenesis of Chronic Obstructive Pulmonary Disease

RATIONALE Neutrophils are key players in chronic obstructive pulmonary disease (COPD), and increased numbers of neutrophils are present in sputum and lung tissue of patients with COPD. Interestingly, immunoglobulin free light chains (IgLC) are able to prolong the life of neutrophils; therefore, IgLC may contribute to the chronic state of inflammation. OBJECTIVES In this study, the relation between IgLC and COPD has been investigated. METHODS We investigated the presence of IgLC in different murine lung emphysema models. IgLC levels in serum from mice and patients with COPD were examined by Western blot analysis and ELISA, respectively. IgLC levels in lung tissue were determined by immunohistochemistry. Fluorescence-activated cell sorter and immunofluorescent analysis were used to detect binding between IgLC and human neutrophils. Interleukin-8 (CXCL8) release by neutrophils after IgLC incubation was measured by ELISA. The effect of F991, an IgLC antagonist, was examined on the neutrophil influx in murine lungs after 5 days of smoke exposure. MEASUREMENTS AND MAIN RESULTS Increased levels of IgLC in serum of cigarette smoke-exposed and cigarette smoke extract-treated mice compared with control mice were observed. Patients with COPD showed increased serum IgLC and expression of IgLC in lung tissue compared with healthy volunteers. Interestingly, IgLC bound to neutrophils and activated neutrophils to release CXCL8. F991 inhibited the IgLC binding to neutrophils and reduced the smoke-induced neutrophil influx in murine lungs after smoke exposure. CONCLUSIONS This study describes for the first time an association between neutrophils and IgLC in the pathophysiology of COPD, which could open new avenues to targeted treatment of this chronic disease.

Immunoglobulin Free Light Chains Are Increased in Hypersensitivity Pneumonitis and Idiopathic Pulmonary Fibrosis

Background Idiopathic pulmonary fibrosis (IPF), a devastating lung disorder of unknown aetiology, and chronic hypersensitivity pneumonitis (HP), a disease provoked by an immunopathologic reaction to inhaled antigens, are two common interstitial lung diseases with uncertain pathogenic mechanisms. Previously, we have shown in other upper and lower airway diseases that immunoglobulin free light chains (FLCs) are increased and may be involved in initiating a local inflammation. In this study we explored if such a mechanism may also apply to HP and IPF. Methods In this study we examined the presence of FLC in serum and BAL fluid from 21 IPF and 22 HP patients and controls. IgG, IgE and tryptase concentrations were measured in BAL fluid only. The presence of FLCs, plasma cells, B cells and mast cells in lung tissue of 3 HP and 3 IPF patients and 1 control was analyzed using immunohistochemistry. Results FLC concentrations in serum and BAL fluid were increased in IPF and HP patients as compared to control subjects. IgG concentrations were only increased in HP patients, whereas IgE concentrations were comparable to controls in both patient groups. FLC-positive cells, B cells, plasma cells, and large numbers of activated mast cells were all detected in the lungs of HP and IPF patients, not in control lung. Conclusion These results show that FLC concentrations are increased in serum and BAL fluid of IPF and HP patients and that FLCs are present within affected lung tissue. This suggests that FLCs may be involved in mediating pathology in both diseases.

Effects of Free Immunoglobulin Light Chains on Viral Myocarditis

Rationale: In recent work, we have demonstrated a crucial role of mast cells in the development of viral myocarditis. Viral infection could lead to increased synthesis of free immunoglobulin light chains (FLC) and our earlier work showed that FLC can trigger mast cell activation. Objective: We studied the possible involvement of FLC in the pathogenesis of viral myocarditis, and therapeutic effects of FLC using an animal model of viral myocarditis. Methods and Results: DBA/2 mice were inoculated intraperitoneally with encephalomyocarditis (EMC) virus. Serum levels and concentrations in the heart of &kgr; FLC on day 14 in mice inoculated with EMC virus were significantly increased compared with controls. Myocardial viral concentration was significantly inhibited, the area of myocardial lesions was smaller in mice treated with &kgr; or &lgr; FLC, and survival of mice given FLC significantly improved. In contrast, an FLC antagonist deteriorated myocarditis. &kgr; and &lgr; FLC chains inhibited EMC viral replication in human amnion cells in vitro. &lgr; FLC significantly increased the gene expression of interleukin-10 in the heart which was previously shown to improve viral myocarditis when given exogenously. FLC also tended to increase the gene expressions of interferon-&agr; and -&ggr; in the heart mice. Conclusions: FLC have antiviral and antiinflammatory effects and improved viral myocarditis in mice. FLC may be promising agents for the treatment of viral myocarditis.