Adhesion to fibronectin via α5ß1 integrin supports expansion of the megakaryocyte lineage in primary myelofibrosis.

Excessive accumulation of extracellular matrix (ECM) is a hallmark of bone marrow (BM) milieu in primary myelofibrosis (PMF). Because cells have the ability to adhere to the surrounding ECM through integrin receptors, we examined the hypothesis that an abnormal ECM-integrin receptor axis contributes to BM megakaryocytosis in JAK2V617F+ PMF. Secretion of ECM protein fibronectin (FN) by BM stromal cells from PMF patients correlates with fibrosis and disease severity. Here, we show that Vav1-hJAK2V617F transgenic mice (JAK2V617F+) have high BM FN content associated with megakaryocytosis and fibrosis. Further, megakaryocytes from JAK2V617F+ mice have increased cell surface expression of the α5 subunit of the α5ß1 integrin, the major FN receptor in megakaryocytes, and augmented adhesion to FN compared with wild-type controls. Reducing adhesion to FN by an inhibitory antibody to the α5 subunit effectively reduces the percentage of CD41+ JAK2V617F+ megakaryocytes in vitro and in vivo. Corroborating our findings in mice, JAK2V617F+ megakaryocytes from patients showed elevated expression of α5 subunit, and a neutralizing antibody to α5 subunit reduced adhesion to FN and megakaryocyte number derived from CD34+ cells. Our findings reveal a previously unappreciated contribution of FN-α5ß1 integrin to megakaryocytosis in JAK2V617F+ PMF.

One-year dietary supplementation with walnuts modifies exosomal miRNA in elderly subjects.

PURPOSE: Epidemiological studies and clinical trials support the association of nut consumption with a lower risk of prevalent non-communicable diseases, particularly cardiovascular disease. However, the molecular mechanisms underlying nut benefits remain to be fully described. MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression and play a pivotal role in health and disease. Exosomes are extracellular vesicles released from cells and mediate intercellular communication. Whether nut consumption modulates circulating miRNAs (c-miRNAs) transported in exosomes is poorly described. METHODS: Cognitively healthy elderly subjects were randomized to either control (n = 110, abstaining from walnuts) or daily supplementation with walnuts (15% of their total energy, ≈30-60 g/day, n = 101) for 1-year. C-miRNAs were screened in exosomes isolated from 10 samples, before and after supplementation, and identified c-miRNA candidates were validated in the whole cohort. In addition, nanoparticle tracking analysis and lipidomics were assessed in pooled exosomes from the whole cohort. RESULTS: Exosomal hsa-miR-32-5p and hsa-miR-29b-3p were consistently induced by walnut consumption. No major changes in exosomal lipids, nanoparticle concentration or size were found. CONCLUSION: Our results provide novel evidence that certain c-miRNAs transported in exosomes are modulated by walnut consumption. The extent to which this finding contributes to the benefits of walnuts deserves further research.

Exosome secretion by eosinophils: A possible role in asthma pathogenesis.

BACKGROUND: Eosinophils secrete several granules that are involved in the propagation of inflammatory responses in patients with pathologies such as asthma. OBJECTIVE: We hypothesized that some of these granules are exosomes, which, when transferred to the recipient cells, could modulate asthma progression. METHODS: Eosinophils were purified from peripheral blood and cultured with or without IFN-γ or eotaxin. Multivesicular bodies (MVBs) in eosinophils were studied by using fluorescence microscopy, transmission electron microscopy (TEM), and flow cytometry. Exosome secretion was measured and exosome characterization was performed with TEM, Western blotting, and NanoSight analysis. RESULTS: Generation of MVBs in eosinophils was confirmed by using fluorescence microscopy and flow cytometry and corroborated by means of TEM. Having established that eosinophils contain MVBs, our aim was to demonstrate that eosinophils secrete exosomes. To do this, we purified exosomes from culture medium of eosinophils and characterized them. Using Western blot analysis, we demonstrated that eosinophils secreted exosomes and that the discharge of exosomes to extracellular media increases after IFN-γ stimulation. We measured exosome size and quantified exosome production from healthy and asthmatic subjects using nanotracking analysis. We found that exosome production was augmented in asthmatic patients. CONCLUSION: Our findings are the first to demonstrate that eosinophils contain functional MVBs and secrete exosomes and that their secretion is increased in asthmatic patients. Thus exosomes might play an important role in the progression of asthma and eventually be considered a biomarker.

SOCS3 silencing attenuates eosinophil functions in asthma patients.

Eosinophils are one of the key inflammatory cells in asthma. Eosinophils can exert a wide variety of actions through expression and secretion of multiple molecules. Previously, we have demonstrated that eosinophils purified from peripheral blood from asthma patients express high levels of suppressor of cytokine signaling 3 (SOCS3). In this article, SOCS3 gene silencing in eosinophils from asthmatics has been carried out to achieve a better understanding of the suppressor function in eosinophils. SOCS3 siRNA treatment drastically reduced SOCS3 expression in eosinophils, leading to an inhibition of the regulatory transcription factors GATA-3 and FoxP3, also interleukin (IL)-10; in turn, an increased STAT3 phosphorilation was observed. Moreover, SOCS3 abrogation in eosinophils produced impaired migration, adhesion and degranulation. Therefore, SOCS3 might be regarded as an important regulator implicated in eosinophil mobilization from the bone marrow to the lungs during the asthmatic process.

Adipocyte-derived exosomes may promote breast cancer progression in type 2 diabetes.

Obesity and metabolic diseases, such as insulin resistance and type 2 diabetes (T2D), are associated with metastatic breast cancer in postmenopausal women. Here, we investigated the critical cellular and molecular factors behind this link. We found that primary human adipocytes shed extracellular vesicles, specifically exosomes, that induced the expression of genes associated with epithelial-to-mesenchymal transition (EMT) and cancer stem­like cell (CSC) traits in cocultured breast cancer cell lines. Transcription of these genes was further increased in cells exposed to exosomes shed from T2D patient­derived adipocytes or insulin-resistant adipocytes and required the epigenetic reader proteins BRD2 and BRD4 in recipient cells. The thrombospondin family protein TSP5, which is associated with cancer, was more abundant in exosomes from T2D or insulin-resistant adipocytes and partially contributed to EMT in recipient cells. Bioinformatic analysis of breast cancer patient tissue showed that greater coexpression of COMP (which encodes TSP5) and BRD2 or BRD3 correlated with poorer prognosis, specifically decreased distant metastasis­free survival. Our findings reveal a mechanism of exosome-mediated cross-talk between metabolically abnormal adipocytes and breast cancer cells that may promote tumor aggressiveness in patients with T2D.

CD9 inhibition reveals a functional connection of extracellular vesicle secretion with mitophagy in melanoma cells.

Tetraspanins are often used as Extracellular Vesicle (EV) detection markers because of their abundance on these secreted vesicles. However, data on their function on EV biogenesis are controversial and compensatory mechanisms often occur upon gene deletion. To overcome this handicap, we have compared the effects of tetraspanin CD9 gene deletion with those elicited by cytopermeable peptides with blocking properties against tetraspanin CD9. Both CD9 peptide or gene deletion reduced the number of early endosomes. CD9 peptide induced an increase in lysosome numbers, while CD9 deletion augmented the number of MVB and EV secretion, probably because of compensatory CD63 expression upregulation. In vivo, CD9 peptide delayed primary tumour cell growth and reduced metastasis size. These effects on cell proliferation were shown to be concomitant with an impairment in mitochondrial quality control. CD9 KO cells were able to compensate the mitochondrial malfunction by increasing total mitochondrial mass reducing mitophagy. Our data thus provide the first evidence for a functional connection of tetraspanin CD9 with mitophagy in melanoma cells.

A new role of diacylglycerol kinase alpha on the secretion of lethal exosomes bearing Fas ligand during activation-induced cell death of T lymphocytes.

Exosomes are small membrane vesicles that intracellularly accumulate into late or multivesicular endosomes (multivesicular bodies, MVB). Exosomes have a particular lipid and protein content, reflecting their origin as intraluminal vesicles of late endosomes. The stimulation of several hematopoietic cells induces the fusion of the limiting membrane of the MVB with the plasma membrane, leading to the release of exosomes towards the extracellular environment. In T lymphocytes, stimulation of the T cell receptor (TCR) induces the fusion of the MVBs with the plasma membrane and exosomes carrying several bio-active proteins are secreted. Among these proteins, the pro-apoptotic protein Fas ligand (FasL) is released as a non-proteolysed form (mFasL), associated to the exosomes. These mFasL-bearing exosomes may trigger the apoptosis of T lymphocytes. Here, we present evidences supporting a role of diacylglycerol kinase alpha (DGKalpha), a diacylglycerol (DAG)-consuming enzyme, on the secretion of exosomes carrying mFasL, and the subsequent activation-induced cell death (AICD) on a T cell line and primary T lymphoblasts.

Exosomes from eosinophils autoregulate and promote eosinophil functions.

Eosinophils are able to secrete exosomes that have an undefined role in asthma pathogenesis. We hypothesized that exosomes released by eosinophils autoregulate and promote eosinophil function. Eosinophils of patients with asthma (n = 58) and healthy volunteers (n = 16) were purified from peripheral blood, and exosomes were isolated and quantified from eosinophils of the asthmatic and healthy populations. Apoptosis, adhesion, adhesion molecules expression, and migration assays were performed with eosinophils in the presence or absence of exosomes from healthy and asthmatic individuals. Reactive oxygen species (ROS) were evaluated by flow cytometry with an intracellular fluorescent probe and nitric oxide (NO) and a colorimetric kit. In addition, exosomal proteins were analyzed by mass spectrometry. Eosinophil-derived exosomes induced an increase in NO and ROS production on eosinophils. Moreover, exosomes could act as a chemotactic factor on eosinophils, and they produced an increase in cell adhesion, giving rise to a specific augmentation of adhesion molecules, such as ICAM-1 and integrin α2. Protein content between exosomes from healthy and asthmatic individuals seems to be similar in both groups. In conclusion, we found that exosomes from the eosinophils of patients with asthma could modify several specific eosinophil functions related to asthma pathogenesis and that they could contribute fundamentally to the development and maintenance of asthma.

Simulated gastrointestinal digestion reduces the allergic reactivity of shrimp extract proteins and tropomyosin.

Shrimp are highly allergenic foods. Current management are limited to the avoidance of foods. Therefore, there is an unmet need for a safe and effective therapy using modified allergens. This study focuses on assessing the potential for modification of the allergenicity of shrimp proteins following heat treatment or simulated gastric digestion. Shrimp proteins do not reduce their IgE reactivity after heat treatment but it is reduced by simulated gastric digestion in a time- and dose-dependent manner. Tropomyosin in shrimp extract is worse digested than purified tropomyosin. After 60 min of 10 U/µg pepsin digestion, a strong inhibition was produced in the in vivo skin reactivity of shrimp extracts and in activation of basophils from allergic patients. Immunisation experiments performed in rabbits demonstrated that digested boiled shrimp extract is able to induce IgG antibodies that block the IgE binding to the untreated boiled shrimp extract in shrimp-allergic patients. Building on our observations, digestion treatment could be an effective method for reducing shrimp allergenicity while maintaining the immunogenicity.

Mitochondrial Respiration Controls Lysosomal Function during Inflammatory T Cell Responses.

The endolysosomal system is critical for the maintenance of cellular homeostasis. However, how endolysosomal compartment is regulated by mitochondrial function is largely unknown. We have generated a mouse model with defective mitochondrial function in CD4(+) T lymphocytes by genetic deletion of the mitochondrial transcription factor A (Tfam). Mitochondrial respiration deficiency impairs lysosome function, promotes p62 and sphingomyelin accumulation, and disrupts endolysosomal trafficking pathways and autophagy, thus linking a primary mitochondrial dysfunction to a lysosomal storage disorder. The impaired lysosome function in Tfam-deficient cells subverts T cell differentiation toward proinflammatory subsets and exacerbates the in vivo inflammatory response. Restoration of NAD(+) levels improves lysosome function and corrects the inflammatory defects in Tfam-deficient T cells. Our results uncover a mechanism by which mitochondria regulate lysosome function to preserve T cell differentiation and effector functions, and identify strategies for intervention in mitochondrial-related diseases.

New shrimp IgE-binding proteins involved in mite-seafood cross-reactivity.

SCOPE: Shrimp is a seafood consumed worldwide and the main cause of severe allergenic reactions to crustaceans. Seafood allergy has been related to mite sensitization, mainly mediated by tropomyosin, but other proteins could be involved. The aim of the study was to identify new shrimp allergens implicated in mite-seafood cross-reactivity (CR) in two different climate populations: dry and humid climates. METHODS AND RESULTS: Shrimp and mite IgE-binding profiles of patients from continental dry and humid climates were analyzed by immunoblotting, and the most frequently recognized Solenocera melantho shrimp proteins were identified by MS as α-actinin, ß-actin, fructose biphosphate aldolase, arginine kinase, sarcoplasmic calcium-binding protein, and ubiquitin. Using inhibition immunoblot assays, we demonstrate that tropomyosin and ubiquitin were responsible for mite-seafood CR from both climates; but also α-actinin and arginine kinase are implicated in dry- and humid-climate populations, respectively. Reciprocal inhibition assays demonstrated that mites are the primary sensitizer in humid-climate, as shrimp is in continental dry-climate population. CONCLUSION: Several new shrimp allergens have been identified and should be considered in the diagnosis and treatment of shrimp allergy and mite-seafood CR. Differences in mite-seafood CR were founded to be based on the climate.

Gene silencing of SOCS3 by siRNA intranasal delivery inhibits asthma phenotype in mice.

Suppresors of cytokine signaling (SOCS) proteins regulate cytokine responses and control immune balance. Several studies have confirmed that SOCS3 is increased in asthmatic patients, and SOCS3 expression is correlated with disease severity. The objective of this study was to evaluate if delivering of SOCS3 short interfering RNA (siRNA) intranasally in lungs could be a good therapeutic approach in an asthma chronic mouse model. Our results showed that intranasal treatment with SOCS3-siRNA led to an improvement in the eosinophil count and the normalization of hyperresponsiveness to methacholine. Concomitantly, this treatment resulted in an improvement in mucus secretion, a reduction in lung collagen, which are prominent features of airway remodeling. The mechanism implies JAK/STAT and RhoA/Rho-kinase signaling pathway, because we found a decreasing in STAT3 phosphorylation status and down regulation of RhoA/Rho-kinase protein expression. These results might lead to a new therapy for the treatment of chronic asthma.

Análisis molecular del gen P53 y microsátelites en muestras endoscópicas de adenomas y carcinomas de colon / Molecular analysis of P53 gene and microsatellites in endoscopic samples of colonic adenomas and carcinomas

Uno de los eventos moleculares que participa en la secuencia adenoma-carcinoma colorrectal es la presencia de mutaciones en el antioncogen p53, cuyo producto proteico regula el ciclo celular. Asimismo, el análisis de microsatélites es un indicador indirecto de inestabilidad génica en tejido tumoral, permitiendo realizar un screening rápido de todo el genoma. En tejidos frescos y mucosa normal de ocho pacientes, se analizaron molecularmente por PCR-SSCP los exones 4 a 8 del gen p53 y microsatélites. Estos datos fueron correlacionados con el diagnóstico morfológico-inmunohistoquímico (IHQ). Fueron observadas alteraciones del gen p53 en el tejido tumoral en tres de ellos; se encontraron mutaciones puntuales en el exón 6 en dos pacientes: uno portador de un adenoma tubular y otro con un adenoma mixto (túbulo-velloso) ambos con displasia de alto grado y p53+IHQ tenía ua mutación en el exón 7. En un adenocarcinoma con sobreexpresión de p53 por IHQ no se detectaron alteraciones de dicho gen. La presencia de mutación sería un indicador de transformación tumoral y corroboraría el seguimiento más estricto en pacientes con lesiones de alto grado. El análisis de microsatélites en los 8 pacientes no determinó la existencia de anomalías génicas.