Extracellular Vesicles as Emerging Players in Intercellular Communication: Relevance in Mast Cell-Mediated Pathophysiology.

Mast cells are major effector cells in eliciting allergic responses. They also play a significant role in establishing innate and adaptive immune responses, as well as in modulating tumor growth. Mast cells can be activated upon engagement of the high-affinity receptor FcεRI with specific IgE to multivalent antigens or in response to several FcεRI-independent mechanisms. Upon stimulation, mast cells secrete various preformed and newly synthesized mediators. Emerging evidence indicates their ability to be a rich source of secreted extracellular vesicles (EVs), including exosomes and microvesicles, which convey biological functions. Mast cell-derived EVs can interact with and affect other cells located nearby or at distant sites and modulate inflammation, allergic response, and tumor progression. Mast cells are also affected by EVs derived from other cells in the immune system or in the tumor microenvironment, which may activate mast cells to release different mediators. In this review, we summarize the latest data regarding the ability of mast cells to release or respond to EVs and their role in allergic responses, inflammation, and tumor progression. Understanding the release, composition, and uptake of EVs by cells located near to or at sites distant from mast cells in a variety of clinical conditions, such as allergic inflammation, mastocytosis, and lung cancer will contribute to developing novel therapeutic approaches.

Lung cancer-derived extracellular vesicles: a possible mediator of mast cell activation in the tumor microenvironment.

Activated mast cells are often found in the tumor microenvironment. They have both pro- and anti-tumorigenic roles, depending on the tumor type. Several lines of evidence suggest that the tumor microenvironment contains multiple soluble factors that can drive mast cell recruitment and activation. However, it is not yet clear how mast cells are activated by tumor cells. In this study, we explored whether tumor-derived microvesicles (TMV) from non-small cell lung cancer (NSCLC) cells interact with human mast cells, activate them to release cytokines, and affect their migratory ability. PKH67-labelled TMV isolated from NSCLC cell lines were found to be internalized by mast cells. This internalization was first noticed after 4 h and peaked within 24 h of co-incubation. Furthermore, internalization of TMV derived from NSCLC cell lines or from surgical lung tissue specimens resulted in ERK phosphorylation, enhanced mast cell migratory ability and increased release of cytokines and chemokines, such as TNF-α and MCP-1. Our data are thus, consistent with the conclusion that TMV have the potential to influence mast cell activity and thereby, affect tumorigenesis.

Characterization of chronic urticaria and associated conditions in a large population of adolescents.

BACKGROUND: Although chronic spontaneous urticaria (CSU) affects all age groups, data regarding CSU in adolescents is scarce. OBJECTIVE: To characterize the epidemiology, demographics, and comorbidities associated with CSU in a large, cross-sectional nationwide population of adolescents. METHODS: Medical records of 16-year-old candidate conscripts to the Israeli Defense Forces were reviewed. Data were collected on the prevalence and severity of CSU, as well as the demographics, medical comorbidities, medication use, and blood test results of affected individuals. RESULTS: Medical records of 1,108,833 consecutive 16-year-old adolescents were reviewed. A total of 6617 (0.6%) adolescents received CSU diagnoses. CSU was increased in female conscripts (odds ratio [OR] 1.13, 95% confidence interval [CI] 1.07-1.19, P < .001) and adolescents with higher socioeconomic scores (OR 1.92, 95% CI 1.56-2.32, P < .001). Individuals with CSU were significantly more likely to have allergic diseases, including food allergy (OR 7.31, 95% CI 6.13-8.72), allergic rhinitis (OR 2.9, 95% CI 2.71-3.11), atopic dermatitis (OR 2.35, 95% CI 2.03-2.72), and asthma (OR 1.46, CI 1.35-1.57). CONCLUSION: Our work provides an account of CSU in a large cohort of adolescents. We found a strong link between CSU and atopic diseases. Further investigation is needed to decipher the mechanism underlying this observed association.

MicroRNA-4443 regulates mast cell activation by T cell-derived microvesicles.

BACKGROUND: The mechanism by which mast cells (MCs) are activated in T cell-mediated inflammatory processes remains elusive. Recently, we have shown that microvesicles derived from activated T cells (mvT*s) can stimulate MCs to degranulate and release several cytokines. OBJECTIVE: The aim of this study was to characterize the contribution of microRNAs (miRs) delivered by microvesicles to MC activation. METHODS: miR profiling was performed with NanoString technology and validated by using real-time PCR. The biological role of mvT* miR was verified by overexpression of miRs in MCs using mimic or inhibitory molecules and analyzing the effect on their predicted targets. RESULTS: mvT*s were found to downregulate the expression of the tyrosine phosphatase protein tyrosine phosphatase receptor type J (PTPRJ), a known extracellular signal-regulated kinase inhibitor. Bioinformatics analysis predicted that miR-4443 regulates the PTPRJ gene expression. Indeed, miR-4443, which was present in mvT*s, was also found to be overexpressed in human MCs stimulated with these MVs. α-Amanitin insensitivity confirmed that overexpression of miR-4443 was not due to transcriptional activation. The luciferase reporter assay indicated that the 3' untranslated region of PTPRJ was targeted by this miR. Transfection of MCs with mimic or inhibitor of miR-4443 resulted in decreased or enhanced PTPRJ expression, respectively. Furthermore, miR-4443 regulated extracellular signal-regulated kinase phosphorylation and IL-8 release in MCs activated by mvT*s. CONCLUSION: These results support a scenario by which T cell-derived microvesicles act as intercellular carriers of functional miR-4443, which might exert heterotypic regulation of PTPRJ gene expression in MCs, leading to their activation in the context of T cell-mediated inflammatory processes.

MicroRNA Involvement in Allergic and Non-Allergic Mast Cell Activation.

Allergic inflammation is accompanied by the coordinated expression of numerous genes and proteins that initiate, sustain, and propagate immune responses and tissue remodeling. MicroRNAs (miRNAs) are a large class of small regulatory molecules that are able to control the translation of target mRNAs and consequently regulate various biological processes at the posttranscriptional level. MiRNA profiles have been identified in multiple allergic inflammatory diseases and in the tumor microenvironment. Mast cells have been found to co-localize within the above conditions. More specifically, in addition to being essential in initiating the allergic response, mast cells play a key role in both innate and adaptive immunity as well as in modulating tumor growth. This review summarizes the possible role of various miRNAs in the above-mentioned processes wherein mast cells have been found to be involved. Understanding the role of miRNAs in mast cell activation and function may serve as an important tool in developing diagnostic as well as therapeutic approaches in mast cell-dependent pathological conditions.

The Involvement of Protein Kinase D in T Cell-Induced Mast Cell Activation.

BACKGROUND: It has recently been reported that mast cells (MC) can be activated to degranulate and release certain cytokines in response to direct physical contact with activated but not resting T cells or their membranes. The MAPK family members ERK and p38 were found to participate. In this work, we further characterize the signaling events involved in this novel pathway of activation. METHODS: Human MC were stimulated by activated T cell membranes (T*m). Phosphorylation of kinases was assessed by Western blotting. Protein kinase D (PKD) translocation was visualized by confocal microscopy. Degranulation was assessed by ß-hexosaminidase release and cytokine production by ELISA. RESULTS: Stimulation of human MC by activated T*m resulted in the activation of PKD. PKD inhibition by the specific pharmacological inhibitor Gö6976 resulted in a reduction in the phosphorylation of p38 but not ERK. Gö6976 also inhibited degranulation and cytokine release. CONCLUSIONS: MC stimulation by physical contact with T cells results in PKD activation, leading to the phosphorylation of p38, degranulation and release of cytokines. Understanding the molecular events associated with T cell-induced MC activation might lead to therapeutic approaches for controlling T cell-mediated inflammatory processes in which MC participate.

T cell-derived microvesicles induce mast cell production of IL-24: relevance to inflammatory skin diseases.

BACKGROUND: It has recently been shown that microvesicles derived from activated T cells can stimulate human mast cells (MCs) to degranulate and release several cytokines. OBJECTIVE: The aim of this study was to characterize microvesicle-induced MC expression patterns. Through identification of unique cytokine and chemokine expression, we attempted to reveal pathogenetic roles for this pathway of MC activation. METHODS: T cell-derived microvesicles were labeled with PKH67 to allow visualization of their interaction with human MCs. Consequent gene expression profiling was studied by using a whole-genome microarray and analyzed for identification of cellular pathway clusters. Expression of 3 selected genes, chemokine (C-C motif) ligand 3 (CCL3), chemokine (C-C motif) ligand 7 (CCL7), and IL24, was validated by means of quantitative RT-PCR and specific ELISA. IL24, which has not been recognized heretofore in MCs, was also tested for its effect on keratinocyte signal transducer and activator of transcription 3 phosphorylation and for its presence in MCs in psoriatic skin lesions. RESULTS: Uptake and internalization of activated T cell-derived microvesicles into human MCs occurred within 24 hours. Microvesicles induced the upregulation of several clusters of genes, notably those that are cytokine related. Among these, IL24 appeared to be a hallmark of microvesicle-induced activation. MC-derived IL-24, in turn, activates keratinocytes in vitro, as manifested by signal transducer and activator of transcription 3 (STAT3) phosphorylation, and is produced in MCs within psoriatic lesions. CONCLUSION: Production of IL-24 is a unique feature of microvesicle-induced MC activation because its production by these cells has not been recognized thus far. We propose that this MC-derived cytokine might contribute to the pathologic findings in T cell-mediated skin inflammation.

Increased Prevalence of Diabetes Mellitus in a Non-Obese Adult Population: HIV-Infected Ethiopians.

BACKGROUND: Diabetes mellitus (DM) is a metabolic sequel in people infected with HIV, especially following the advent of HAART. This may be a particular concern in immigrants due to lifestyle changes. OBJECTIVES: To characterize the prevalence of DM in HIV-infected Ethiopians in Israel, and to define the risk factors. METHODS: We retrospectively screened the records of 173 HIV-infected Ethiopians and 69 HIV-infected non-Ethiopian HIV patients currently registered at the HIV Clinic of Meir Medical Center. Data were also retrieved from 1323 non-HIV Ethiopians treated in the hospital between 2007 and 2012. The presence of DM was determined by family physician diagnosis as recorded in the hospital database or by the presence of one or more of the following: fasting glucose > 127 mg/dl, hA1C > 6.5% (> 48 mmol/mol), or blood glucose > 200 mg/dl. Population data and risk factors for DM were analyzed by univariate and multivariate analyses. RESULTS: Among HIV-infected Ethiopian subjects, the prevalence of DM was 31% (54/173) compared to 4% (3/69) in HIV-infected non-Ethiopians and 8% (102/1323) in non-HIV-infected Ethiopians (P < 0.0001). The relatively increased prevalence of DM was age independent, but most noticeable in those under the median age (< 42 years). Body mass index (BMI) was a predictor for DM (OR 1.263, CI 1.104-1.444, P = 0.001), although its values did not vary between the two ethnic groups. CONCLUSIONS: HIV-infected Ethiopians are more likely to develop DM at low BMI values compared to non-Ethiopians. This observation questions the relevance of accepted BMI values in this population and suggests that preventive measures against DM be routinely taken in these subjects.

[NON-CLONAL MAST CELL ACTIVATION SYNDROME: DESCRIPTION OF SERIES OF PATIENTS, DIAGNOSTIC CRITERIA AND TREATMENT].

A non-clonal mast cell activation syndrome is a newly emerged diagnosis. It shares the clinical features of anaphylaxis and mastocytosis despite having distinct mast cell biology. In this,paper we describe a series of patients representing the spectrum of non-clonal mast cell activation syndrome (nc-MCAS). We highlight the clinical manifestations of nc-MCAS as well as the diagnostic criteria and treatment options.

Allergic manifestations in women with silicone breast implants.

INTRODUCTION: Silicone breast implants (SBI) result in immune dysregulation and are associated with autoimmune diseases. Recently, we reported dysregulated levels of IgG autoantibodies directed against G protein-coupled receptors (GPCRs) of the autonomic nervous system which were linked to the autoimmune dysautonomia in silicone breast implant illness (SBII). AIMS: We aimed to explore the possible association between allergy with dysregulated IgE autoantibodies directed against GPCRs of the autonomic nervous system in women with SBI. METHODS: Circulating levels of IgE autoantibodies against GPCRs of the autonomic nervous system (adrenergic, muscarinic, endothelin and angiotensin receptors) have been evaluated in women with SBIs who complained of allergic symptoms, and compared to subjects with SBI without allergic manifestations and to age-matched healthy women without SBI. RESULTS: We report a significant dysregulation in three circulating autoantibodies: IgE-beta1 adrenergic receptor (B1AR), IgE-alpha 1 adrenergic receptor (A1AR) and IgE-muscarinic acetylcholine receptor type 1 (M1R) autoantibodies in women with SBI who complained of allergic symptoms. CONCLUSIONS: Allergic reactions associated with SBI are not uncommon. Imbalance of circulating levels of IgE autoantibodies against GPCRs of the autonomic nervous system might play a role not only in allergic reactions, but also in other enigmatic aspects of SBII such as autoimmune dysautonomia.

Increased eosinophilic responses in splenectomized patients.

BACKGROUND: The spleen is a key organ within the immune system. Its removal is known to bring about adverse effects such as an increased susceptibility to overwhelming infection. Few reports have suggested that the spleen may play a role in controlling eosinophilic responses, mostly based on animal models. OBJECTIVES: To examine whether the human spleen impacts eosinophil numbers in the blood. METHODS: We have retrospectively analyzed eosinophil counts and medical records of 29 patients who had undergone splenectomy between 2000 and 2010. Statistical comparison was performed between post-splenectomy blood counts and both pre-splenectomy and control values. Data regarding the clinical settings around hypereosinophilia events were obtained from patient charts. RESULTS: An increased rate of eosinophilia was observed after splenectomy as compared with normal individuals. Furthermore, a considerable proportion of patients who had undergone splenectomies (8/29) presented peak eosinophil numbers exceeding 1,000/mm(3), reaching a maximum of 3,070/mm(3). These values were mostly encountered perioperatively or during episodes of acute infection. CONCLUSIONS: Our data indicate that impaired control of eosinophilic responses is a long-term post-splenectomy effect and is evident in the context of acute stress. We suggest that the spleen plays a significant role in controlling eosinophil levels and that these cells may mediate some of the harmful consequences observed after removal of the spleen.

T cell-induced mast cell activation: a role for microparticles released from activated T cells.

Close physical proximity between mast cells and T cells has been demonstrated in several T cell-mediated inflammatory processes. However, the way by which mast cells are activated in these T cell-mediated immune responses has not been fully elucidated. We previously identified and characterized a novel mast cell activation pathway initiated by physical contact with activated T cells and showed that this pathway is associated with degranulation and cytokine release. In this study, we provide evidence that mast cells may also be activated by microparticles released from activated T cells that are considered miniature versions of a cell. Microparticles were isolated from supernatants of activated T cells by Centricon filtration or by high-speed centrifugation and identified by electron microscopy, flow cytometry (Annexin stain), and expression of the integrin LFA-1. Stimulated T cells were found to generate microparticles that induce degranulation and cytokine (IL-8 and oncostatin M) release from human mast cells. Mast cell activation by T cell microparticles involved the MAPK signaling pathway. The results were similar when mast cells were stimulated by activated fixed T cells or by whole membranes of the latter. This suggests that microparticles carry mast cell-activating factors similar to cells from which they originate. By releasing microparticles, T cells might convey surface molecules similar to those involved in the activation of mast cells by cellular contact. By extension, microparticles might affect the activity of mast cells, which are usually not in direct contact with T cells at the inflammatory site.

Activation of mast cells by trimeric G protein Gi3; coupling to the A3 adenosine receptor directly and upon T cell contact.

Mast cells are key players in mediating and amplifying allergic and inflammatory reactions. Previously, we identified the G-protein, Gi3, as the cellular target of receptor mimetic basic secretagogues that activate mast cell independently of IgE. In this study, we demonstrate that Gi3 is the cellular target of the adenosine A3 receptor (A3R), a G-protein coupled receptor involved in inflammation and the pathophysiology of asthma. By using a cell permeable peptide comprising the C-terminal end of Galphai3 fused to an importation sequence (ALL1) as a selective inhibitor of Gi3 signaling, we show that by coupling to Gi3, the A3R stimulates multiple signaling pathways in human mast cells, leading to upregulation of cytokines, chemokines, and growth factors. We further show that after contact with activated T cell membranes, endogenous adenosine binds to and activates the A3R, resulting in Gi3-mediated signaling. Specifically, the majority of ERK1/2 signaling initiated by contact with activated T cell membranes, is mediated by Gi3, giving rise to ALL1-inhibitable cellular responses. These results unveil the physiological G-protein coupled receptor that couples to Gi3 and establish the important role played by this G-protein in inflammatory conditions that involve adenosine-activated mast cells.

Tumor-Derived Extracellular Vesicles Induce CCL18 Production by Mast Cells: A Possible Link to Angiogenesis.

Mast cells (MCs) function as a component of the tumor microenvironment (TME) and have both pro- and anti-tumorigenic roles depending on the tumor type and its developmental stage. Several reports indicate the involvement of MCs in angiogenesis in the TME by releasing angiogenic mediators. Tumor cells and other cells in the TME may interact by releasing extracellular vesicles (EVs) that affect the cells in the region. We have previously shown that tumor-derived microvesicles (TMVs) from non-small-cell lung cancer (NSCLC) cells interact with human MCs and activate them to release several cytokines and chemokines. In the present study, we characterized the MC expression of other mediators after exposure to TMVs derived from NSCLC. Whole-genome expression profiling disclosed the production of several chemokines, including CC chemokine ligand 18 (CCL18). This chemokine is expressed in various types of cancer, and was found to be associated with extensive angiogenesis, both in vitro and in vivo. We now show that CCL18 secreted from MCs activated by NSCLC-TMVs increased the migration of human umbilical cord endothelial cells (HUVECs), tube formation and endothelial- to-mesenchymal transition (EndMT), thus promoting angiogenesis. Our findings support the conclusion that TMVs have the potential to influence MC activity and may affect angiogenesis in the TME.

Mast cell activation by fibrinogen-related homologous c-terminal peptides (haptides) modulates systemic blood pressure.

BACKGROUND: Haptides are a family of short peptides homologous to C-termini sequences of fibrinogen chains ß and γ (haptides Cß and preCγ, respectively) which were previously shown to penetrate and bind cells. OBJECTIVES: This work investigates the systemic effect of the haptides with possible clinical implications. METHODS: Intra-arterial monitoring in rats recorded the haptides' effects on systemic blood pressure. In parallel, their effect was also tested in vitro on isolated rat peritoneal mast cells and on human mast cells. RESULTS: Intra-arterial monitoring in rats showed that intravenous administration of low haptides concentrations (35-560 µg/kg rat) caused a shocklike behavior with transient decrease in the systolic and diastolic blood pressure by up to 55% (P < .05) in a dose-dependent manner and a minor increase in their heart rate. Randomly scrambled sequences of the haptides had no such effect, suggesting a specific interaction with receptors. Intravenous administration of blockers to histamine receptors H1 and H2 before haptides administration attenuated this effect. Furthermore, in vitro incubation of human LAD2 mast cell line or isolated rat peritoneal mast cells with the haptides caused degranulation of the mast cells. We found that the haptides Cß and preCγ activated mast cells causing histamine release, resulting in a steep decrease in blood pressure, comparable to anaphylactic shock. CONCLUSION: In treating vascular occlusive diseases, massive fibrinolysis is induced, and haptide-containing sequences are released. We suggest that treatment with histamine receptor blockers or with mast cell stabilizing agents in such pathological conditions may overcome this effect.

Cellular therapy in 2010: focus on autoimmune and cardiac diseases.

Stem cell therapy has developed extensively in recent years, leading to several new clinical fields. The use of mesenchymal stromal cells sparks special interest, as it reveals the importance of the paracrine and immunomodulatory effects of these supporting cells, in disease and in cure. This review discusses our current understanding of the basic clinical principles of stem cell therapy and demonstrates the broad range of this treatment modality by examining two relatively new therapeutic niches--autoimmune and cardiac diseases.

Neurologic manifestations as presenting symptoms of endocarditis.

The features of infective endocarditis include both cardiac and non-cardiac manifestations. Neurologic complications are seen in up to 40% of patients with infective endocarditis and are the presenting symptom in a substantial percentage. We describe in detail the clinical scenarios of three patients admitted to our hospital, compare their characteristics and review the recent literature describing neurologic manifestations of infective endocarditis. Our patients demonstrate that infective endocarditis can develop without comorbidity or a valvular defect. Moreover, our patients were young and lacked the most common symptom of endocarditis: fever. The most common neurologic manifestations were focal neurologic deficits and confusion. We conclude that infective endocarditis should always be considered in patients presenting with new-onset neurologic complaints, especially in those without comorbidities or other risk factors. A prompt diagnosis should be reached and antibiotic treatment initiated as soon as possible.

Human mast cells release oncostatin M on contact with activated T cells: possible biologic relevance.

BACKGROUND: We have recently demonstrated that mast cells can be activated by heterotypic adhesion to activated T cells. OBJECTIVE: We sought to perform gene expression profiling on human mast cells activated by either IgE cross-linking or by T cells and to characterize one of the cytokines, oncostatin M (OSM). METHODS: Gene expression profiling was done by means of microarray analysis, OSM expression was validated by means of RT-PCR, and the product was measured by means of ELISA in both the LAD 2 human mast cell line and in cord blood-derived human mast cells. Immunocytochemistry was used to localize OSM in human mast cells, and its biologic activity was verified by its effect on the proliferation of human lung fibroblasts. RESULTS: OSM was expressed and released specifically on T cell-induced mast cell activation but not on IgE cross-linking. OSM was localized to the cytoplasm, and its expression was inhibited by dexamethasone and mitogen-activated protein kinase inhibitors. OSM was also found to be biologically active in inducing lung fibroblast proliferation that was partially but significantly inhibited by anti-OSM mAb. In vivo mast cells were found to express OSM in both biopsy specimens and bronchoalveolar lavage fluid from patients with sarcoidosis. CONCLUSION: The production of OSM by human mast cells might represent one link between T cell-induced mast cell activation and the development of a spectrum of structural changes in T cell-mediated inflammatory processes in which mast cells have been found to be involved.

MOR: a simulation-based assessment centre for evaluating the personal and interpersonal qualities of medical school candidates.

CONTEXT: Medical school admissions traditionally rely heavily on cognitive variables, with non-cognitive measures assessed through interviews only. In recognition of the unsatisfactory reliability and validity of traditional interviews, medical schools are increasingly exploring alternative approaches that can provide improved measures of candidates' personal and interpersonal qualities. METHODS: An innovative assessment centre (MOR [Hebrew acronym for 'selection for medicine']) was designed to measure candidates' personal and interpersonal attributes. Three assessment tools were developed: behavioural stations, including encounters with simulated patients and group tasks; an autobiographical questionnaire, and a judgement and decision-making questionnaire. Candidates were evaluated by trained raters on four qualities: interpersonal communication; ability to handle stress; initiative and responsibility, and self-awareness. RESULTS: In the years 2004-05, the 588 medical school candidates with the highest cognitive scores were tested; this resulted in a change of approximately 20% in the cohort of accepted students compared with previous admission criteria. Internal consistency ranged from 0.80 to 0.88; inter-rater reliability ranged from 0.62 to 0.77 for the behavioural stations and from 0.72 to 0.95 for the questionnaires; test-retest score correlation was 0.7. The correlation between candidates' MOR scores and cognitive scores approached zero, reflecting the value of MOR in the screening process. Feedback from participants indicated that MOR was perceived as fair and appropriate for medical school screening. DISCUSSION: MOR is a reliable tool for measuring non-cognitive attributes in medical school candidates. It has high content and face validity. Furthermore, its implementation conveys the importance of maintaining humanist characteristics in the medical profession to students and faculty staff.