Exosomal CD63 in critically ill patients with sepsis.

CD63 is one of the tetraspanin protein family members that is ubiquitously expressed on exosomes and is involved in the signal transduction of various types of immune cells. It may thus contribute to immunometabolic mechanisms of cellular and organ dysfunction in sepsis. Nonetheless, the association of exosomal CD63 with the severity and mortality of sepsis is not well known. Therefore, in the present study, the overall levels of exosomal CD63 were evaluated to ascertain whether they were associated with organ failure and mortality in patients with sepsis. Exosomal CD63 was measured from prospectively enrolled critically-ill patients with sepsis (n = 217) and healthy control (n = 20). To detect and quantify exosomes in plasma, a commercially available enzyme-linked immunosorbent assay kit was used according to the manufacturer's protocol. The total number of exosomal CD63 was determined by quantifying the immunoreactive CD63. The association between plasma levels of exosomal CD63 and sequential organ failure assessment (SOFA) score was assessed by a linear regression method. The best cut-off level of exosomal CD63 for 28-day mortality prediction was determined by Youden's index. Among 217 patients with sepsis, 143 (66%) patients were diagnosed with septic shock. Trends of increased exosomal CD63 levels were observed in control, sepsis, and septic-shock groups (6.6 µg/mL vs. 42 µg/mL vs. 90 µg/mL, p < 0.001). A positive correlation between exosomal CD63 and SOFA scores was observed in patients with sepsis (r value = 0.35). When patients were divided into two groups according to the best cut-off level, the group with higher exosomal CD63 levels (more than 126 µg/mL) was significantly associated with 28-day and in-hospital mortality. Moreover, the Kaplan-Meier survival method showed a significant difference in 90-day survival between patients with high- and low-exosomal CD63 levels (log-rank p = 0.005). Elevated levels of exosomal CD63 were associated with the severity of organ failure and predictive of mortality in critically ill patients with sepsis.

Resistance Training Diminishes the Expression of Exosome CD63 Protein without Modification of Plasma miR-146a-5p and cfDNA in the Elderly.

Aging-associated inflammation is characterized by senescent cell-mediated secretion of high levels of inflammatory mediators, such as microRNA (miR)-146a. Moreover, a rise of circulating cell-free DNA (cfDNA) is also related to systemic inflammation and frailty in the elderly. Exosome-mediated cell-to-cell communication is fundamental in cellular senescence and aging. The plasma changes in exercise-promoted miR-146a-5p, cfDNA, and exosome release could be the key to facilitate intercellular communication and systemic adaptations to exercise in aging. Thirty-eight elderly subjects (28 trained and 10 controls) volunteered in an 8-week resistance training protocol. The levels of plasma miR-146a-5p, cfDNA, and exosome markers (CD9, CD14, CD63, CD81, Flotillin [Flot]-1, and VDAC1) were measured prior to and following training. Results showed no changes in plasma miR-146a-5p and cfDNA levels with training. The levels of exosome markers (Flot-1, CD9, and CD81) as well as exosome-carried proteins (CD14 and VDAC1) remained unchanged, whereas an attenuated CD63 response was found in the trained group compared to the controls. These findings might partially support the anti-inflammatory effect of resistance training in the elderly as evidenced by the diminishment of exosome CD63 protein expression, without modification of plasma miR-146a-5p and cfDNA.

A light-up fluorescence resonance energy transfer magnetic aptamer-sensor for ultra-sensitive lung cancer exosome detection.

In vitro liquid biopsy based on exosomes offers promising opportunities for fast and reliable detection of lung cancers. In this work, we present a fluorescence resonance energy transfer (FRET) magnetic aptamer-sensor for magnetic enrichment of exosomes with aptamers and detection of cancerous-surface proteins based on a light-up FRET strategy. Fluorescent quantum dots (QDs) and aptamers were introduced onto magnetic nanoparticles and the fluorescence emission turned down when the aptamers were paired with their complementary DNA on the surface of Au nanoparticles. Later, competitive binding of exosomes with the aptamers expelled the Au nanoparticles resulting in an exosome concentration-dependent linear increase of QD fluorescence intensity in a broad exosome concentration range (5 × 102-5 × 109 particles per mL). As found in our work, this system behaved ultra-sensitively and the calculated detection limit of this FRET magnetic aptamer-sensor was as low as 13 particles per mL. Furthermore, taking epithelial cancer-specific antigen (epithelial cell adhesion molecule, EpCAM) screening as a typical example, our built FRET magnetic aptamer-sensor allowed a rapid and efficient distinction of all the epithelial cancer cases (7 lung cancers and 5 other cancers) from health volunteers with 100% accuracy.

High serum extracellular vesicle miR-10b expression predicts poor prognosis in patients with acute myeloid leukemia.

BACKGROUND: Increasing evidence have demonstrated that serum extracellular vesicle microRNAs (EV-miRNAs) are promising noninvasive biomarkers for various cancer types. OBJECTIVE: In this study, we aimed to investigate and evaluate the potential clinical significance of serum EV-miR-10b for acute myeloid leukemia (AML). METHODS: Blood samples were collected from a cohort of 95 de novo AML patients and 80 healthy individuals. Of all AML patients, 51 patients were considered as cytogenetic normal AML (CN-AML). Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) was performed to measure the expression levels of serum EV-miR-10b. RESULTS: The extracellular vesicles we extracted from the serum samples were positive for EV/exosome markers including TSG101, CD63, Flotillin-1 and CD9. The expression levels of serum EV-miR-10b were significantly higher in AML/CN-AML patients than healthy controls. In addition, serum EV-miR-10b expression was strongly correlated with aggressive clinical characteristics. Moreover, receiver operating characteristic analysis showed serum EV-miR-10b yielded an area under the curve of 0.875, with 77.89% specificity and 82.50% sensitivity in identifying AML patients from normal controls. Furthermore, AML patients with higher serum EV-miR-10b expression had significantly shorter survival and serum EV-miR-10b was demonstrated to be an independent prognostic marker for overall survival in AML. CONCLUSIONS: Taken together, serum EV-miR-10b might serve as a promising biomarker for predicting the prognosis of AML.

The CD63 basophil activation test as a diagnostic tool for assessing autoimmunity in patients with chronic spontaneous urticaria.

BACKGROUND: Over the past years, it has become widely recognized that a proportion of chronic spontaneous urticaria (CSU) cases is of autoimmune origin, however, the search for reliable diagnostic tools to confirm underlying autoimmune pathophysiology is ongoing. The CD63 basophil activation test (CD63 BAT) has recently become useful for diagnosing autoimmune CSU. OBJECTIVES: To analyse the correlation between positive CD63 BAT results, total IgE antibody levels, and the presence of autoimmune thyroiditis as a comorbidity in patients diagnosed with CSU. MATERIALS AND METHODS: We performed a retrospective analysis of CD63 BAT results obtained from 87 CSU and 20 non-CSU patients. The information extracted from the patients' records included age, gender, total IgE levels, clinical history of autoimmune thyroiditis (AT), and the presence of anti-thyroid autoantibodies. RESULTS: Positive CD63 BAT results were significantly more frequent in CSU patients compared with non-CSU subjects (p=0.045). Furthermore, we found a strong significant negative correlation between the stimulation index (SI) value for CD63 BAT and total IgE levels in CD63 BAT-positive CSU patients (p=0.004; Spearman's rank correlation coefficient ρ=-0.322), meaning that higher SI values corresponded to lower total IgE values, and vice versa. CONCLUSION: The current standard set of diagnostic tools cannot be reliably used to determine when CSU is caused by autoimmune mechanisms. There is evidence that CD63 BAT represents a helpful diagnostic tool for detecting underlying autoimmunity. We show that high SI values in CD63 BAT-positive CSU patients correlate negatively with their total IgE levels. The clinical relevance of this effect needs to be investigated further.

Prostate cancer sheds the αvß3 integrin in vivo through exosomes.

The αvß3 integrin has been shown to promote aggressive phenotypes in many types of cancers, including prostate cancer. We show that GFP-labeled αvß3 derived from cancer cells circulates in the blood and is detected in distant lesions in NOD scid gamma (NSG) mice. We, therefore, hypothesized that αvß3 travels through exosomes and tested its levels in pools of vesicles, which we designate extracellular vesicles highly enriched in exosomes (ExVs), and in exosomes isolated from the plasma of prostate cancer patients. Here, we show that the αvß3 integrin is found in patient blood exosomes purified by sucrose or iodixanol density gradients. In addition, we provide evidence that the αvß3 integrin is transferred through ExVs isolated from prostate cancer patient plasma to ß3-negative recipient cells. We also demonstrate the intracellular localization of ß3-GFP transferred via cancer cell-derived ExVs. We show that the ExVs present in plasma from prostate cancer patients contain higher levels of αvß3 and CD9 as compared to plasma ExVs from age-matched subjects who are not affected by cancer. Furthermore, using PSMA antibody-bead mediated immunocapture, we show that the αvß3 integrin is expressed in a subset of exosomes characterized by PSMA, CD9, CD63, and an epithelial-specific marker, Trop-2. Finally, we present evidence that the levels of αvß3, CD63, and CD9 remain unaltered in ExVs isolated from the blood of prostate cancer patients treated with enzalutamide. Our results suggest that detecting exosomal αvß3 integrin in prostate cancer patients could be a clinically useful and non-invasive biomarker to follow prostate cancer progression. Moreover, the ability of αvß3 integrin to be transferred from ExVs to recipient cells provides a strong rationale for further investigating the role of αvß3 integrin in the pathogenesis of prostate cancer and as a potential therapeutic target.

The Aging Process Alters IL-1ß and CD63 Levels Differently in Extracellular Vesicles Obtained from the Plasma and Cerebrospinal Fluid.

OBJECTIVE(S): The aim of this study was to investigate exosomal markers and inflammatory cargo of extracellular vesicles (EVs) obtained from cerebrospinal fluid (CSF) and plasma in the aging process. We also studied the inflammatory cargo by quantifying IL-1ß levels. METHODS: Male Wistar rats, aged 3 and 21 months, were used (n = 12 in each group). The CSF and plasma of animals were collected, and isolation of EVs was performed using a commercial kit. Total protein concentration, acetylcholinesterase (AChE) activity, and CD63 and IL-1ß levels were evaluated. RESULTS: AChE activity in EVs increased in both samples, specifically in the circulating EVs and those in the CSF of the older group. An age-related increase was observed in CD63 levels in EVs from the CSF but a decrease was observed in plasma EVs of the older group. Student's t test showed that the young adult rats had significantly higher circulating IL-1ß levels in the EVs compared to the aged ones, without any effect on central content. CONCLUSION: Our data suggest that the normal aging process causes different changes in the profiles of central and circulating EVs. Altered IL-1ß levels in circulating EVs can be linked, at least partly, to age-related inflammatory conditions, and a disruption of the CFS exosomes in aged rats, evaluated by CD63 levels, can be related to susceptibility to neurodegenerative disorders.

Prevention of venous thromboembolism after resection of primary liver cancer with low molecular weight heparin and its association with P-selectin, lysosomal granule glycoprotein, platelet activating factor and plasma D-dimer.

OBJECTIVE: We aimed at analyzing the efficacy of low molecular heparin in the prevention of venous thromboembolism (VTE) after resection of primary liver cancer and at exploring the correlation of VTE with P-selectin (CD62P), lysosomal granule glycoprotein (CD63), platelet activating factor (PAF) and plasma D-dimer (D-D). PATIENTS AND METHODS: A total of 233 patients treated with primary liver cancer resection in our hospital from February 2014 to October 2016 were enrolled in this study. The patients were randomly divided into the observation group (n=117) and the control group (n=116). The observation group received a subcutaneous injection of low molecular weight heparin at 2-7 days after surgery, and the control group was not treated with anticoagulation. The prevalence of VTE and the changes of CD62P, CD63, PAF, and D-D before and after treatment were compared between the two groups. The VTE prevalence after surgery, the changes of CD62P, CD63, PAF, D-D before and after surgery and the correlation of the above indexes with VTE were analyzed. RESULTS: The prevalence of VTE in the observation group was 0.85% (1/117), which was lower than that of the control group (13.79%) (16/116); the difference was statistically significant (p<0.05). There was no significant difference in blood coagulation function between the two groups before and after operation (p>0.05). The CD62P, CD63, PAF, and D-D of the two groups were continuously decreased after the operation, and the serum CD62P, CD63 and plasma D-D of the observation group 6 d and 10 d after operation were lower than that of the control group; the difference was statistically significant (p<0.05). The serum CD62P, CD63 and plasma D-D in the VTE group 6 d and 10 d after operation were lower than those in the non-VTE group; the differences were statistically significant (p<0.05). CONCLUSIONS: Low molecular weight heparin can effectively prevent VTE after primary liver cancer resection. Regularly monitoring CD62P, CD63, PAF, and D-D in patients after the operation is pivotal for early diagnosis, evaluation and treatment of VTE.

Basophil Activation Test Application in Drug Hypersensitivity Diagnosis: An Empirical Approach.

BACKGROUND: The diagnosis of drug hypersensitivity reactions (DHRs) is based both on clinical history and in vivo tests, such as specific IgE and cutaneous tests, when available. OBJECTIVES: The aim of this work was to evaluate the basophil activation test (BAT) as a supplementary tool for drug challenges and drug allergy diagnosis. METHOD: We evaluated 204 outpatients reporting DHRs. Available serum-specific IgE drugs were determined and cutaneous tests were performed when appropriate. BAT was performed immediately after blood sampling. The expression of CD63 was evaluated with flow cytometry. The test was considered positive when CD63 expression was > 5% and the stimulation index (the ratio of the percentage of CD63-expressing cells with drug exposure/percentage of CD63-expressing cells with wash buffer) was > 2. Patients who reported mild to severe reactions and those with a discrepancy between clinical history and BAT underwent a challenge test. RESULTS: The drugs that caused adverse reactions were mainly antibiotics (49%). Non-steroid anti-inflammatory drugs (NSAID) were cited as responsible for DHRs in 37%, with the remaining 14% being due to other drugs. BAT revealed a high specificity (92%) and low sensitivity for antibiotics (40%). For the suspected reactions to penicillin, both the in vitro tests supported 94% of the diagnoses. We also observed a high specificity in the case of challenge with NSAIDs (100% specificity). CONCLUSIONS: BAT is effective in discriminating adverse drug reactions, whilst only more critical cases require integrated evaluations and more complex clinical examinations. It is relevant that the concordance of anamnesis and in vitro tests reduce the need for challenge testing, limiting them to selected cases.

Circulating extracellular vesicles in the aging process: impact of aerobic exercise.

Our aim was to investigate transitory and delayed exercise effects on serum extracellular vesicles (EVs) in aging process. Male Wistar rats of 3-, 21-, and 26-month old were allocated into exercised and sedentary groups. The exercise protocol consisted in a daily moderate treadmill exercise (20 min daily during 2 weeks). Trunk blood was collected 1 and 18 h after the last exercise session, and circulating EVs were obtained. CD63 levels and acetylcholinesterase (AChE) activity were used as markers of exosome, a subtype of EVs. In addition, the quantification of amyloid-ß (Aß) levels and the oxidative status parameters, specifically reactive species content, superoxide dismutase (SOD) activity, and SOD1 content were evaluated. Aged rats showed reduced CD63 levels and increased AChE activity in circulating exosomes compared to young ones. Moreover, higher reactive species levels were found in circulating EVs of aged rats. Delayed exercise effects were observed on peripheral EVs, since CD63, reactive species content, and AChE activity were altered 18 h after the last exercise session. Our results suggest that the healthy aging process can modify circulating EVs profile, and exercise-induced beneficial effects may be related to its modulation on EVs.

Exosome enrichment by ultracentrifugation and size exclusion chromatography.

Exosomes are a subset of extracellular vesicles (EVs) that have important roles in intercellular communication. They contain and carry bioactive molecules within their membranes which are delivered to target cells. Reproducible isolation and enrichment of these exosomes will aid in evaluation of cellular communication. We present an approach that involved the pre-processing of plasma, combined with ultracentrifugation (UC) and size exclusion chromatography (SEC) to isolate EVs and subsequently enrich exosomes. Four variations of this approach (denoted methods I to IV) were compared. Coupling an ultracentrifugation method with size exclusion chromatography (Method II) provided the best yield by nanoparticle tracking analyses (NTA), the presence of the exosomal markers CD63, Flotillin-1 and TSG-101 (immunoblotting) and showed exosome morphology using transmission electron microscopy (TEM). This method provides an efficient way to enrich the exosomes from blood (plasma), which could be potentially employed for clinical diagnostic assessment and therapeutic intervention.

Identification of promising plasma immune biomarkers to differentiate active pulmonary tuberculosis.

Although much research has been done related to biomarker discovery for tuberculosis infection, a set of biomarkers that can discriminate between active and latent TB diseases remains elusive. In the current study we correlate clinical aspects of TB disease with changes in the immune response as determined by biomarkers detected in plasma. Our study measured 18 molecules in human plasma in 17 patients with active disease (APTB), 14 individuals with latent tuberculosis infection (LTBI) and 16 uninfected controls (CTRL). We found that active tuberculosis patients have increased plasma levels of IL-6, IP-10, TNF-α, sCD163 and sCD14. Statistical analysis of these biomarkers indicated that simultaneous measurement of sCD14 and IL-6 was able to diagnose active tuberculosis infection with 83% accuracy. We also demonstrated that TNF-α and sCD163 were correlated with tuberculosis severity. We showed that the simultaneous detection of both plasma sCD14 and IL-6 is a promising diagnostic approach to identify APTB, and further, measurement of TNF-α and sCD163 can identify the most severe cases of tuberculosis.

Effects of DHA-enriched fish oil on monocyte/macrophage activation marker sCD163, asymmetric dimethyl arginine, and insulin resistance in type 2 diabetic patients.

BACKGROUND: The beneficial effects of n-3 polyunsaturated fatty acids on reducing cardiovascular risks are well documented. However, the relative effect on some markers of macrophage activation and vascular function is unclear. OBJECTIVE: The primary objective of this study was to investigate the effects of docosahexaenoic acid (DHA)-enriched fish oil on the marker of monocyte/macrophage activation factor soluble CD163, asymmetric dimethyl arginine (ADMA), and insulin resistance in type 2 diabetic patients. METHODS: In this double-blind randomized controlled trial, 72 type 2 diabetic patients with an age between 30-70 years and body mass index (BMI) of 18.5 to 40 kg/m(2) were randomly assigned to receive 2.4-g DHA-enriched fish oil or placebo per day for 8 weeks. Anthropometric measurements, biochemical, and body composition analyses were assessed at baseline and end of study. Analysis of covariance (ANCOVA) was conducted by controlling for possible confounders to assess between-group differences. RESULTS: Serum levels of sCD163, triglycerides, waist circumference (WC), and weight to height ratio (WHtR) decreased significantly in the fish oil group when compared with the control group. Serum ADMA concentration decreased in the fish oil group with no significant between-group differences. Controlling for confounders revealed that the differences observed in sCD163, triglycerides, WC, and WHtR remained statistically significant. CONCLUSIONS: Short-time fish oil supplementation decreased serum sCD163, triglycerides levels, WC, and WHtR in T2DM patients. Because of the positive relationship between sCD163 levels and some T2DM and obesity-related complications, it seems that DHA can be considered as a key intervention in obesity and T2DM.

Quantification of plasma exosome is a potential prognostic marker for esophageal squamous cell carcinoma.

Exosomes play important roles in cancer progression. Although its contents (e.g., proteins and microRNAs) have been focused on in cancer research, particularly as potential diagnostic markers, the exosome behavior and methods for exosome quantification remain unclear. In the present study, we analyzed the tumor-derived exosome behavior and assessed the quantification of exosomes in patient plasma as a biomarker for esophageal squamous cell carcinoma (ESCC). A CD63-GFP expressing human ESCC cell line (TE2-CD63-GFP) was made by transfection, and mouse subcutaneous tumor models were established. Fluorescence imaging was performed on tumors and plasma exosomes harvested from mice. GFP-positive small vesicles were confirmed in the plasma obtained from TE2-CD63-GFP tumor-bearing mice. Patient plasma was collected in Chiba University Hospital (n=86). Exosomes were extracted from 100 µl of the plasma and quantified by acetylcholinesterase (AChE) activity. The relationship between exosome quantification and the patient clinical characteristics was assessed. The quantification of exosomes isolated from the patient plasma revealed that esophageal cancer patients (n=66) expressed higher exosome levels than non-malignant patients (n=20) (P=0.0002). Although there was no correlation between the tumor progression and the exosome levels, exosome number was the independent prognostic marker and low levels of exosome predicted a poor prognosis (P=0.03). In conclusion, exosome levels may be useful as an independent prognostic factor for ESCC patients.

Generation of a novel transgenic rat model for tracing extracellular vesicles in body fluids.

Extracellular vesicles (EVs) play an important role in the transfer of biomolecules between cells. To elucidate the intercellular transfer fate of EVs in vivo, we generated a new transgenic (Tg) rat model using green fluorescent protein (GFP)-tagged human CD63. CD63 protein is highly enriched on EV membranes via trafficking into late endosomes and is often used as an EV marker. The new Tg rat line in which human CD63-GFP is under control of the CAG promoter exhibited high expression of GFP in various body tissues. Exogenous human CD63-GFP was detected on EVs isolated from three body fluids of the Tg rats: blood serum, breast milk and amniotic fluid. In vitro culture allowed transfer of serum-derived CD63-GFP EVs into recipient rat embryonic fibroblasts, where the EVs localized in endocytic organelles. These results suggested that this Tg rat model should provide significant information for understanding the intercellular transfer and/or mother-child transfer of EVs in vivo.

Inhibition of platelet function with clopidogrel is associated with a reduction of inflammation in patients with peripheral artery disease.

BACKGROUND: The reactivity of platelets is increased in patients with peripheral artery disease (PAD). RANTES and sCD40L are chemokines which are stored in the alpha-granules of platelets. Clopidogrel inhibits and thus reduces platelet reactivity. Whether a treatment with clopidogrel is associated with an inhibition of systemic inflammation in patients with PAD has not been thoroughly explored. This study examined the effect of clopidogrel on platelet reactivation and the release of inflammatory chemokines in patients with PAD. METHODS: 40 patients with PAD were randomized into two groups. In the first group A the patients were treated with 100mg acetylsalicylic acid (ASA) and additional placebo for 4weeks. The patients in group B received 75mg/d clopidogrel in addition to ASA 100mg for 4weeks. After obtaining blood at days 0, 7 and 28 the platelet activation was determined by measuring the surface protein expression of CD63, CD62p and thrombospondin (TSP) after stimulation with TRAP and ADP. The release of the chemokines RANTES and sCD40L from platelets was analyzed by ELISA. RESULTS: Platelet activation markers (CD62p and CD63) and chemokine RANTES were significantly reduced in patients with PAD after 7 and 28days after treatment with clopidogrel. No alterations were found in TSP expression and sCD40L during the treatment. CONCLUSION: The treatment with clopidogrel leads to a reduction of platelet reactivity and release of RANTES from the platelets of patients with PAD.

Impact of preeclampsia on megakaryocytopoesis and platelet homeostasis of preterm infants.

UNLABELLED: The aim of this article is to investigate the megakaryopoyesis and thrombopoiesis in preterm infants born to mothers with preeclampsia and the potential effects mediated by soluble fms-like tyrosine kinase 1 (sFlt1) and thrombopoietin (TPO). A perspective case-control study was performed on 26 cord blood of preterm newborns born to mothers with preeclampsia (PE group) and 26 of preterms born to mothers without preeclampsia (control group). Circulating megakaryocyte count and megakaryocyte colony forming units (CFU-MK) were quantified by whole blood infiltration method and plasma clot culture system, respectively. Platelet activation markers, CD62P and CD63, were estimated by flow cytometry. Immunosorbent assays (ELISA) were employed to estimate plasma levels of sFlt1 and TPO of the two groups. When compared to the controls, infants born to mothers with PE had significantly lower peripheral platelet count (PE vs. CONTROLS: 157.9 [44.6] vs. 239.6 [57.5] × 10(9)/l, p < 0.001), circulating MK count (5.8 [1.0] vs. 7.7 [0.9]/ml, p < 0.001) and CFU-MK (14.1 [2.1] vs. 20.1 [2.8]/1 × 10(5) cell, p < 0.001); greater expressions of CD62P (15.5 [2.3] vs. 11.4 [1.9]% platelets, p < 0.001) and CD63 (12.3 [2.4] vs. 9.0 [1.6]% platelets, p < 0.001); increased plasma Flt level (130.1 [8.0] vs. 97.7 [8.7] pg/ml, p < 0.001) and TPO level (129.5 [17.8] vs. 98.9 [11.8] pg/ml, p < 0.001). In PE group, sFlt instead of TPO showed a significantly negative relationship with platelet counts, CFU-MK and circulating MK count, a positive relationship with CD62P, CD63 expressions. In control group, both sFlt and TPO did not show any relationship with these parameters. sFlt played important role in megakaryocytopoesis and platelet homeostasis in preterm infants born to mothers with PE. Its mechanism maybe the effect of impaired megakaryocyte formation and increased platelet activation.

Telomeric repeat-containing RNA (TERRA) constitutes a nucleoprotein component of extracellular inflammatory exosomes.

Telomeric repeat-containing RNA (TERRA) has been identified as a telomere-associated regulator of chromosome end protection. Here, we report that TERRA can also be found in extracellular fractions that stimulate innate immune signaling. We identified extracellular forms of TERRA in mouse tumor and embryonic brain tissue, as well as in human tissue culture cell lines using RNA in situ hybridization. RNA-seq analyses revealed TERRA to be among the most highly represented transcripts in extracellular fractions derived from both normal and cancer patient blood plasma. Cell-free TERRA (cfTERRA) could be isolated from the exosome fractions derived from human lymphoblastoid cell line (LCL) culture media. cfTERRA is a shorter form (∼200 nt) of cellular TERRA and copurifies with CD63- and CD83-positive exosome vesicles that could be visualized by cyro-electron microscopy. These fractions were also enriched for histone proteins that physically associate with TERRA in extracellular ChIP assays. Incubation of cfTERRA-containing exosomes with peripheral blood mononuclear cells stimulated transcription of several inflammatory cytokine genes, including TNFα, IL6, and C-X-C chemokine 10 (CXCL10) Exosomes engineered with elevated TERRA or liposomes with synthetic TERRA further stimulated inflammatory cytokines, suggesting that exosome-associated TERRA augments innate immune signaling. These findings imply a previously unidentified extrinsic function for TERRA and a mechanism of communication between telomeres and innate immune signals in tissue and tumor microenvironments.

A Potential Mechanism of High-Dose Ticagrelor in Modulating Platelet Activity and Atherosclerosis Mediated by Thymic Stromal Lymphopoietin Receptor.

Abnormal expression of thymic stromal lymphopoietin (TSLP) and its receptor (TSLPR) was found in patients with acute coronary syndrome. Ticagrelor, an oral platelet ADP P2Y12 receptor antagonist, is widely used in these patients. The aim of this study was to verify whether different doses of ticagrelor regulated plaque progression and platelet activity by modulating TSLP/TSLPR. Seventy-five ApoE-/- mice were randomly divided into five groups: (1) high-cholesterol diet (HCD, n = 15); (2) HCD plus ticagrelor 25 mg/kg/d (T1, n = 15); (3) HCD plus ticagrelor 50 mg/kg/d (T2, n = 15); (4) HCD plus ticagrelor 100 mg/kg/d (T3, n = 15); and (5) a normal diet group (ND, n = 15). At day 0 and at week 16, blood lipids and serum TSLP levels, expression of TSLPR, CD62, and CD63, platelet aggregation, platelet ATP release, PI3K/Akt signaling pathway, and plaque morphology were assessed. HCD increased TSLPR expression and atherosclerosis progression but high-dose ticagrelor (100 mg/kg) moderated this trend. TSLPR was positively correlated with Akt1, platelet aggregation, corrected plaque area, and vulnerability index in the T3 group (P<0.01). In conclusion, low-dose ticagrelor only inhibited platelet activity. Besides this inhibition, high-dose ticagrelor modulated platelet activity and atherosclerosis mediated by TSLPR, potentially through the PI3K/Akt signal pathway.

Increased Levels of Sphingosylphosphorylcholine (SPC) in Plasma of Metabolic Syndrome Patients.

Recent developments in lipid mass spectrometry enable extensive lipid class and species analysis in metabolic disorders such as diabesity and metabolic syndrome. The minor plasma lipid class sphingosylphosphorylcholine (SPC) was identified as a ligand for lipid sensitive G-protein coupled receptors playing a key role in cell growth, differentiation, motility, calcium signaling, tissue remodeling, vascular diseases and cancer. However, information about its role in diabesity patients is sparse. In this study, we analyzed plasma lipid species in patients at risk for diabesity and the metabolic syndrome and compared them with healthy controls. Our data show that SPC is significantly increased in plasma samples from metabolic syndrome patients but not in plasma from patients at risk for diabesity. Detailed SPC species analysis showed that the observed increase is due to a significant increase in all detected SPC subspecies. Moreover, a strong positive correlation is observed between total SPC and individual SPC species with both body mass index and the acute phase low grade inflammation marker soluble CD163 (sCD163). Collectively, our study provides new information on SPC plasma levels in metabolic syndrome and suggests new avenues for investigation.