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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., (© 2021. The Author(s).)

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 × 10 2 -5 × 10 9 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.

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.

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.

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.

The αvβ3 integrin has been shown to promote aggressive phenotypes in many types of cancers, including prostate cancer. We show that GFP-labeled αvβ3 derived from cancer cells circulates in the blood and is detected in distant lesions in NOD scid gamma (NSG) mice. We, therefore, hypothesized that αvβ3 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 αvβ3 integrin is found in patient blood exosomes purified by sucrose or iodixanol density gradients. In addition, we provide evidence that the αvβ3 integrin is transferred through ExVs isolated from prostate cancer patient plasma to β3-negative recipient cells. We also demonstrate the intracellular localization of β3-GFP transferred via cancer cell-derived ExVs. We show that the ExVs present in plasma from prostate cancer patients contain higher levels of αvβ3 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 αvβ3 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 αvβ3, CD63, and CD9 remain unaltered in ExVs isolated from the blood of prostate cancer patients treated with enzalutamide. Our results suggest that detecting exosomal αvβ3 integrin in prostate cancer patients could be a clinically useful and non-invasive biomarker to follow prostate cancer progression. Moreover, the ability of αvβ3 integrin to be transferred from ExVs to recipient cells provides a strong rationale for further investigating the role of αvβ3 integrin in the pathogenesis of prostate cancer and as a potential therapeutic target., (Copyright © 2018 Elsevier B.V. All rights reserved.)

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.

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-β (Aβ) 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.

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.

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-1β 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-1β 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-1β 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-1β 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., (© 2018 S. Karger AG, Basel.)

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., (© 2018 S. Karger AG, Basel.)

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., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

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.

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.

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., (Copyright © 2016 National Lipid Association. Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2016 Elsevier Inc. All rights reserved.)

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) 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.

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.

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.

Background: Adverse systemic reactions (SRs) are more common in honeybee venom immunotherapy (VIT) than in wasp VIT. Factors that might be associated with SRs during the honeybee VIT are poorly understood., Objective: Our aim was to evaluate risk factors for SRs during the build-up phase of honeybee venom immunotherapy., Methods: We included 93 patients who underwent ultra-rush honeybee VIT. The adverse SRs and their severity was compared to various immunological (sIgE, tIgE, basophil CD63 response, baseline tryptase, and skin tests), patient-specific (age, sex, cardiovascular conditions and medications, and other allergic diseases), and sting-specific factors (anaphylaxis severity, time interval to onset of symptoms, and absence of cutaneous symptoms)., Results: Twenty-three patients (24.7%) experienced mild SRs and 13 patients (14%) severe SRs. In five patients with severe SRs, the build-up was stopped. High basophil allergen sensitivity, evaluated as dose-response curve metrics of EC15, EC50, CD-sens, AUC, or the response to submaximal 0.01 μg/mL of venom concentration, was the most significant risk factor and only independent predictor of severe SRs and/or build-up stop. Time interval of <5 min after sting to onset of symptoms and lower specific IgEs to rApi m1 was also associated with severe SRs. There was no difference in other immunological, patient-specific, or sting-specific factors, including the baseline tryptase. None of the studied factors was associated with mild SRs., Conclusion and Clinical Relevance: High basophil allergen CD63 sensitivity phenotype was a major indicator of severe adverse SRs during the build-up phase of honeybee VIT. Possibly role was also showed for short latency to filed sting reaction and low sIgE to rApi m1. Before honeybee VIT, measurement of basophil allergen sensitivity should be used to identify patients with a high risk for severe side-effects., (© 2015 John Wiley & Sons Ltd.)

Background: Platelet secretion is critical to development of acute thrombotic occlusion. Platelet dense granules contain a variety of important hemostatically active substances. Nevertheless, biogenesis of platelet granules is poorly understood., Objectives: Serum- and glucocorticoid-inducible kinase 1 (SGK1) has been shown to be highly expressed in platelets and megakaryocytes, but its role in the regulation of platelet granule biogenesis and its impact on thrombosis has not been investigated so far., Methods and Results: Electron microscopy analysis of the platelet ultrastructure revealed a significant reduction in the number and packing of dense granules in platelets lacking SGK1 (sgk1(-/-) ). In sgk1(-/-) platelets serotonin content was significantly reduced and activation-dependent secretion of ATP, serotonin and CD63 significantly impaired. In vivo adhesion after carotis ligation was significantly decreased in platelets lacking SGK1 and occlusive thrombus formation after FeCl3 -induced vascular injury was significantly diminished in sgk1(-/-) mice. Transcript levels and protein abundance of dense granule biogenesis regulating GTPase Rab27b were significantly reduced in sgk1(-/-) platelets without affecting Rab27b mRNA stability. In MEG-01 cells transfection with constitutively active (S422) (D) SGK1 but not with inactive (K127) (N) SGK1 significantly enhanced Rab27b mRNA levels. Sgk1(-/-) megakaryocytes show significantly reduced expression of Rab27b and serotonin/CD63 levels compared with sgk1(+/+) megakaryocytes. Proteome analysis identified nine further vesicular transport proteins regulated by SGK1, which may have an impact on impaired platelet granule biogenesis in sgk1(-/-) platelets independent of Rab27b., Conclusions: The present observations identify SGK1 as a novel powerful regulator of platelet dense granule biogenesis, platelet secretion and thrombus formation. SGK1 is at least partially effective because it regulates transcription of Rab27b in megakaryocytes., (© 2015 International Society on Thrombosis and Haemostasis.)

Background: Platelet (PLT) concentrates can be cryopreserved in dimethyl sulfoxide (DMSO) and stored at -80°C for 2 years. These storage conditions improve availability in both rural and military environments. Previous phenotypic and in vitro studies of cryopreserved PLTs are limited by comparison to fresh liquid-stored PLTs, rather than PLTs stored over their clinically relevant shelf life. Further, nothing is known of the effect of reconstituting cryopreserved PLTs in plasma stored at a variety of clinically relevant temperatures., Study Design and Methods: Apheresis PLTs were either stored at room temperature for 5 days or cryopreserved at -80°C with 5% DMSO. Cryopreserved PLTs were thawed at 37°C and reconstituted in plasma (stored at different temperatures) and compared to fresh and expired liquid-stored PLTs. In vitro assays were performed to assess glycoprotein expression, PLT activity, microparticle content, and function., Results: Compared to liquid PLTs over storage, cryopreserved PLTs had reduced expression of the key glycoprotein receptors GPIbα and GPIIb. However, the proportion of PLTs expressing activation markers CD62P and CD63 was similar between cryopreserved and liquid-stored PLTs at expiry. Cryopreserved PLT components contained significantly higher numbers of phosphatidylserine- and tissue factor-positive microparticles than liquid-stored PLTs, and these microparticles reduced the time to clot formation and increased thrombin generation., Conclusion: There are distinct differences between cryopreserved and liquid-stored PLTs. Cryopreserved PLTs also have an enhanced hemostatic activity. Knowledge of these in vitro differences will be essential to understanding the outcomes of a clinical trial comparing cryopreserved PLTs and liquid PLTs stored for various durations., (© 2014 AABB.)

Exposure to diesel exhaust is an important cardiovascular risk factor and may promote atherothrombotic events. Some data suggest that polluted air exposure could affect haemostasis through platelet activation. The aim of the study was to investigate the effects of acute exposure to diesel exhaust on platelet activation and platelet function. We tested the hypothesis in a randomised, crossover study in 25 healthy men exposed to ambient and polluted air; 11 of the subjects also performed exercise during exposure sessions. Platelet activation was evaluated by surface expression of CD62P (P-selectin) and CD63 (dense granule glycoprotein) using flow cytometry of labelled platelets. Platelet function was measured using the PFA-100 platelet function analyser and by Multiplate whole blood impedance platelet aggregometry. Acute diesel exhaust exposure had no effect on platelet activation at rest, but exercise in polluted air increased the collagen-induced expression of CD62P and CD63 (both p< 0.05). The increase in the expression of CD62P and CD63 was related to the total amount of PM2.5 inhaled during the exercise sessions (r=+0.58 and +0.60, respectively, both p< 0.05). Platelet aggregation was not impaired after polluted air exposure at rest or during exercise. In conclusion, in healthy subjects, diesel exhaust exposure induces platelet activation as illustrated by a dose-response increase in the release of CD62P and CD63. This platelet priming effect could be a contributor to the triggering of atherothrombotic events related to air pollution exposure.

Background: The diagnosis of immediate allergy is based on clinical data, skin prick tests (SPT), and measurements of allergen-specific IgE (sIgE). Basophil activation test (BAT) can supplement these methods and obviate their disadvantages, and possibly replace allergen challenge tests, such as a nasal provocative test (NPT). In this study, we assessed the influence of different storage times on BAT results. Futhermore, we compared the results of SPT, sIgE and NPT against BAT for common aeroallergens., Methods: BAT was performed in twelve patients with allergic rhinitis sensitized to birch pollen or mites 1, 4 and 24 hours after blood sampling. CD63 was used as an activation marker. Three serial 10-fold dilutions (1:1, 1:10, 100) of allergen extract were employed. The further 10 individuals allergic to mites undergone complete diagnostic evaluation including SPT, sIgE measurements, NPT and BAT. Receiver operating characteristic (ROC) curves were used to compare the diagnostic techniques and tests conditions., Results: Basophil activation expressed as stimulation index did not decline significantly up to 24h. Exposure to causal allergens resulted in a dose-dependent increase in expression of CD63 on peripheral blood basophils in tested individuals. We did not observed substantial differences in results of the investigated diagnostic methods determined by a ROC analysis., Conclusions: Flow-assisted diagnosis of common respiratory allergy relies upon allergen-induced activation of blood basophils can be a useful approach to determine the clinically relevant allergen in sensitized individuals. BAT with inhaled allergens can be performed within 24 hours after blood collecting into a tube with EDTA. Allergen suitable for NPT in appropriate dilutions is a good reagent for use in BAT.

The hemodialysis procedure involves contact between peripheral blood and the surface of dialyzer membranes, which may lead to alterations in the pathways of innate and adaptive immunity. We aimed to study the effect of blood-membrane interaction on human peripheral basophils and neutrophils in hemodialysis with high- and low-permeability polysulfone dialyzers. The surface expression of CD203c (basophil selection marker) and CD63 (activation marker) after activation by the bacterial peptide formyl-methionyl-leucyl-phenylalanine (fMLP) or anti-Fcε receptor I (FcεRI) antibody and the absolute number of basophils was investigated before and after hemodialysis with each of the dialyzers. Moreover, the expression on neutrophils of CD11b, the CD11b active epitope, and CD88 was analyzed in the same groups of individuals. The expression of CD63 in basophils following activation by fMLP was significantly higher in the patient group compared with that in healthy controls, but no differences were observed after activation by anti-FcεRI. During the hemodialysis procedure, the low-flux membrane induced up-regulation of CD63 expression on basophils, while passage through the high-flux membrane did not significantly alter the responsiveness. In addition, the absolute number of basophils was unchanged after hemodialysis with either of the dialyzers and compared with healthy controls. We found no significant differences in the expression of the neutrophil activation markers (CD11b, the active epitope of CD11b, and CD88) comparing the two different dialyzers before and after dialysis and healthy controls. Together, these findings suggest that alterations in basophil activity may be a useful marker of membrane bioincompatibility in hemodialysis., (© 2014 The Authors. Artificial Organs published by The International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.)

Severe dengue is characterised by thrombocytopenia, plasma leakage and bleeding. Platelets are important for preservation of endothelial integrity. We hypothesised that platelet activation with secondary platelet dysfunction contribute to plasma leakage. In adult Indonesian patients with acute dengue, we measured platelet activation status and the response to the platelet agonist TRAP using flow cytometer-based assays. Patients were monitored daily for plasma leakage by ultrasonography. Acute dengue was associated with platelet activation with an increased expression of the activated fibrinogen receptor (αIIbβ3), the lysosomal marker CD63 and the alpha-granule marker CD62P (P-selectin). Upon maximal platelet activation by TRAP, platelet function defects were observed with a significantly reduced maximal activated αIIbβ3 and CD63 expression and reduced platelet-monocyte and platelet-neutrophil complexes. Patients in the lowest tertile of activated αIIbβ3 and CD63 expression had an odds ratio for plasma leakage of 5.2 (95% confidence interval [CI] 1.3-22.7) and 3.9 (95% CI 1.1-13.7), respectively, compared to the highest tertile. Platelet-derived serotonin has previously been related to plasma leakage and we found increased intra-platelet serotonin concentrations in our patients. In conclusion, platelet activation with platelet function alterations can be found in patients with acute dengue and this may contribute to dengue-associated plasma leakage.

Exosomes are membrane vesicles 30-120 nm in diameter that are released by many cell types and carry a cargo of proteins, lipids, mRNA, and microRNA. Cultured adipocytes reportedly release exosomes that may play a role in cell-to-cell communication during the development of metabolic diseases. However, the characteristics and function of exosomes released from adipocytes in vivo remain to be elucidated. Clearly, adipocyte-derived exosomes could exist in the circulation and may be associated with adipocyte-specific proteins such as adipocytokines. We isolated exosomes from serum of mice by differential centrifugation and analyzed adiponectin, leptin, and resistin in the exosome fraction. Western blotting detected adiponectin but no leptin and only trace amounts of resistin in the exosome fraction. The adiponectin signal in the exosome fraction was decreased by proteinase K treatment and completely quenched by a combination of proteinase K and Triton X-100. Quantitative ELISA showed that the exosome fraction contains considerable amounts of adiponectin, but not leptin or resistin. The concentration of adiponectin in the serum and the ratio of adiponectin to total protein in the exosome fraction were lower in obese mice than in lean mice. These results suggest that a portion of adiponectin exists as a transmembrane protein in the exosomes in mouse serum. We propose adiponectin as a marker of exosomes released from adipocytes in vivo., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background: Mild platelet function disorders (PFDs) are complex and difficult to diagnose. The current gold standard test, light transmission aggregometry (LTA), including lumi-aggregometry, is time and labour intensive and blood samples must be processed within a limited time after venepuncture. Furthermore, many subjects with suspected PFDs do not show a platelet abnormality on LTA., Objective: To assess the diagnostic potential of an easy-to-use remote platelet function test (RPFT) as a diagnostic pre-test for suspected PFDs., Methods: A remote platelet function test was compared with lumi-aggregometry in participants recruited to the Genotyping and Phenotyping of Platelets Study (GAPP, ISRCTN 77951167). For the RPFT, whole blood was stimulated with platelet agonists, stabilized with PAMFix and returned to the central laboratory for analysis of P-selectin and CD63 by flow cytometry., Results: For the 61 study participants (42 index cases and 19 relatives) there was a good agreement between lumi-aggregometry and the RPFT, with diagnosis being concordant in 84% of cases (κ = 0.668, P < 0.0001). According to both tests, 29 participants were identified to have a deficiency in platelet function and 22 participants appeared normal. There were four participants where lumi-aggregometry revealed a defect but the RPFT did not, and six participants where the RPFT detected an abnormal platelet response that was not identified by lumi-aggregometry., Conclusion: This study suggests that the RPFT could be an easy-to-use pre-test to select which participants with bleeding disorders would benefit from extensive platelet phenotyping. Further development and evaluation of the test are warranted in a wider population of patients with excessive bleeding and could provide informative screening tests for PFDs., (© 2014 International Society on Thrombosis and Haemostasis.)

Background: Cerebral microembolic signals (MES) may predict increased stroke risk in carotid stenosis. However, the relationship between platelet counts or platelet activation status and MES in symptomatic vs. asymptomatic carotid stenosis has not been comprehensively assessed., Setting: University teaching hospitals., Methods: This prospective, pilot observational study assessed platelet counts and platelet activation status, and the relationship between platelet activation and MES in asymptomatic vs. early (≤ 4 weeks after TIA/stroke) and late phase (≥ 3 months) symptomatic moderate or severe (≥ 50%) carotid stenosis patients. Full blood count measurements were performed, and whole blood flow cytometry was used to quantify platelet surface activation marker expression (CD62P and CD63) and circulating leucocyte-platelet complexes. Bilateral simultaneous transcranial Doppler ultrasound monitoring of the middle cerebral arteries was performed for 1 h to classify patients as MES positive or MES negative., Results: Data from 31 asymptomatic patients were compared with 46 symptomatic patients in the early phase, and 35 of these patients were followed up to the late phase after symptom onset. The median platelet count (211 vs. 200 × 10(9)  L(-1) ; P = 0.03) and the median percentage of lymphocyte-platelet complexes was higher in early symptomatic than asymptomatic patients (2.8 vs. 2.4%; P = 0.001). The percentage of lymphocyte-platelet complexes was higher in early symptomatic than in asymptomatic patients with ≥ 70% carotid stenosis (P = 0.0005) and symptomatic patients recruited within 7 days of symptom onset (P = 0.028). Complete TCD data were available in 25 asymptomatic, 31 early phase symptomatic and 27 late phase symptomatic patients. Twelve per cent of asymptomatic vs. 32% of early phase symptomatic (P = 0.02) and 19% of late phase symptomatic patients (P = 0.2) were MES positive. Early symptomatic MES-negative patients had a higher percentage of lymphocyte-platelet complexes than asymptomatic MES-negative patients (2.8 vs. 2.3%; P = 0.0085)., Discussion: Recently, symptomatic carotid stenosis patients have had higher platelet counts (potentially reflecting increased platelet production, mobilization or reduced clearance) and platelet activation status than asymptomatic patients. MES were more frequently detected in early symptomatic than asymptomatic patients, but the differences between late symptomatic and asymptomatic groups were not significant. Increased lymphocyte-platelet complex formation in recently symptomatic vs. asymptomatic MES-negative patients indicates enhanced platelet activation in this early symptomatic subgroup. Platelet biomarkers, in combination with TCD, have the potential to aid risk-stratification in asymptomatic and symptomatic carotid stenosis patients., (© 2013 International Society on Thrombosis and Haemostasis.)

Background Information: Exosomes are small RNA- and protein-containing extracellular vesicles (EVs) that are thought to mediate hetero- and homotypic intercellular communication between normal and malignant cells.Tumour-derived exosomes are believed to promote re-programming of the tumour-associated stroma to favour tumour growth and metastasis. Currently, exosomes have been intensively studied in carcinomas. However, little is known about their existence and possible role in sarcomas., Results: Here, we report on the identification of vesicles with exosomal features derived from Ewing's sarcoma(ES), the second most common soft-tissue or bone cancer in children and adolescents. ES cell line-derived EV shave been isolated by ultracentrifugation and analysed by flow-cytometric assessment of the exosome-associated proteins CD63 and CD81 as well as by electron microscopy. They proved to contain ES-specific transcripts including EWS-FLI1, which were suitable for the sensitive detection of ES cell line-derived exosomes by qRT-PCRin a pre-clinical model for patient plasma. Microarray analysis of ES cell line-derived exosomes revealed that they share a common transcriptional signature potentially involved in G-protein-coupled signalling, neurotransmitter signalling and stemness., Conclusions: In summary, our results imply that ES-derived exosomes could eventually serve as biomarkers for minimal residual disease diagnostics in peripheral blood and prompt further investigation of their potential biological role in modification of the ES-associated microenvironment, (© 2013 Société Française des Microscopies and Société de Biologie Cellulaire de France. Published by John Wiley & Sons Ltd.)

Exosomes biomarkers mediating important biological process, especially in the systemic disease diagnostics and therapeutics, yet the protective exosomal vesicle structure hinders rapid, simple detection of the harbored molecules. We have established a new method, the electric field-induced release and measurement (EFIRM), which can simultaneously disrupt exosomes to release the contents and on-site monitoring the harbored exosomal RNA/proteins biomarkers. When exposed to a non-uniform electrical field, exosomal RNA and proteins are rapidly released. Bio-recognition of these biomolecules is carried out concurrently. We tested the hypothesis that the lung cancer cell line, H460 stably transfected with hCD63-GFP, would shed hCD63-GFP expressing exosomes that could be detected in serum and saliva. We confirmed in vivo that H460-CD63-GFP shed exosomes were transported to blood and saliva. This result demonstrates for the first time tumor-shed exosomes were detected in saliva, in addition to blood, presenting a new translational utility of exosome-based biomarker detection in saliva., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Aim: A major concern of stent implantation after percutaneous coronary intervention (PCI) is acute stent thrombosis. Effective inhibition of periprocedural platelet function in patients with coronary artery disease (CAD) leads to an improved outcome. In this study, we examined the periprocedural platelet reactivity after administrating bivalirudin during PCI compared to unfractionated heparin (UFH) administration. Further, the effect of bivalirudin on induced tissue factor (TF) expression in smooth muscle cells (SMC) was determined., Methods: Patients with CAD (n = 58) and double antithrombotic medication were treated intraprocedural with UFH (n = 30) or bivalirudin (n = 28). Platelet activation markers were flow cytometrically measured before and after stenting. The expression of TF in SMC was determined by real-time PCR and Western blotting. The thrombogenicity of platelet-derived microparticles and SMC was assessed via a TF activity assay., Results: Bivalirudin significantly diminished the agonist-induced platelet reactivity post-PCI. Compared to UFH treatment, the adenosine diphosphate (ADP) and thrombin receptor-activating peptide (TRAP)-induced thrombospondin expression post-PCI was reduced when bivalirudin was administrated during intervention. In contrast to UFH, bivalirudin reduced the P-selectin expression of unstimulated and ADP-induced platelets post-PCI. Moreover, bivalirudin inhibited the thrombin-, but not FVIIa- or FVIIa/FX-induced TF expression and pro-coagulant TF activity of SMC. Moreover, bivalirudin reduced the TF activity of platelet-derived microparticles postinduction with TRAP or ADP., Conclusions: Bivalirudin is better than UFH in reducing periprocedural platelet activation. Moreover, thrombin-induced TF expression is inhibited by bivalirudin. Thus, bivalirudin seems to be a better anticoagulant during PCI than UFH., (© 2011 Blackwell Publishing Ltd.)

Background: Although vascular-calcification mechanisms are only partially understood, the role of circulating calcifying cells and non-collagenous bone matrix proteins in the bone-vascular axis is emerging. In spite of the fact that platelets represent a cellular interface between hemostasis, inflammation and atherosclerosis, and have a myeloid precursor, a possible involvement in the modulation of vascular calcification has rarely been investigated. We investigated if osteocalcin (OC) is released by platelets and described OC expression in patients with carotid artery occlusive disease., Methods: Expression and release of OC were determined by Western blot, immunofluorescence, fluorescence-activated cell sorting (FACS) and ELISA in human resting and activated platelets and megakaryocytes. Co-localization of platelet aggregates, macrophages, OC and calcifications was studied in carotid endarterectomy specimens and normal tissues., Results: Human platelets expressed OC and co-localized with CD63 in δ-granules. Upon activation with an endogenous mechanism, platelets released OC in the extracellular medium. Expression of OC in megakaryocytes suggested lineage specificity. The OC count in circulating platelets and the released amount were significantly higher in patients with carotid artery occlusive disease than in healthy controls (P < 0.0001) in spite of similar serum levels. In atherosclerotic plaques, OC strongly overlapped with CD41+ platelets in the early stage of calcification, but this was not seen in normal tissues. CD68+OC+ cells were present at the periphery of the calcified zone., Conclusions: Given the active role played by platelets in the atherosclerotic process, the involvement of OC release from platelets in atherosclerotic lesions and the impact of genetic and cardiovascular risk factors in mediating bone-marrow preconditioning should be investigated further., (© 2012 International Society on Thrombosis and Haemostasis.)

Background: A reproducible standard for a graded allergen response in allergic rhinitis is lacking. The aim was to evaluate basophil allergen threshold sensitivity, CD-sens, as a diagnostic complement to nasal allergen challenge., Methods: Twenty-six patients with a history of allergic rhinitis due to grass pollen were intranasally challenged and nasal symptom score and peak nasal inspiratory flow (PNIF) changes were determined after 15 min. A 20% decrease in PNIF or a symptom score ≥2 were considered a positive test. A blood sample for CD-sens was drawn before each challenge. Eighteen patients were tested twice., Results: CD-sens agreed with the positive or negative nasal symptom score in 22/26 and PNIF in 24/26 patients. After the second challenge, 14/18 patients had the same symptom, 17/18 the same PNIF, while all had identical CD-sens classification., Conclusion: CD-sens appears to be a reproducible test for diagnosis of allergic rhinitis with great advantages also for follow-up of disease development and treatment effects., (Copyright © 2012 S. Karger AG, Basel.)

Platelets are currently acknowledged as cells of innate immunity and inflammation and play a complex role in sepsis. We examined whether different types of LPS have different effects on the release of soluble signaling/effective molecules from platelets. We used platelet-rich plasma from healthy volunteers and LPS from two strains of gram-negative bacteria with disparate LPS structures. We combined LPS-stimulated platelet supernatants with reporter cells and measured the PBMC cytokine secretion profiles. Upon stimulation of platelets with both Escherichia coli O111 and Salmonella minnesota LPS, the platelet LPS::TLR4 interaction activated pathways to trigger the production of a large number of molecules. The different platelet supernatants caused differential PBMC secretion of IL-6, TNFα, and IL-8. Our data demonstrate that platelets have the capacity to sense external signals differentially through a single type of pathogen recognition receptor and adjust the innate immune response appropriately for pathogens exhibiting different types of 'danger' signals., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Objective: Staphylococcus aureus can induce platelet aggregation. The rapidity and degree of this correlates with the severity of disseminated intravascular coagulation, and depends on platelet peptidoglycans. Surface-located thiol isomerases play an important role in platelet activation. The staphylococcal extracellular adherence protein (Eap) functions as an adhesin for host plasma proteins. Therefore we tested the effect of Eap on platelets., Methods and Results: We found a strong stimulation of the platelet-surface thiol isomerases protein disulfide isomerase and endoplasmic reticulum stress proteins 57 and 72 by Eap. Eap induced thiol isomerase-dependent glycoprotein IIb/IIIa activation, granule secretion, and platelet aggregation. Treatment of platelets with thiol blockers, bacitracin, and anti-protein disulfide isomerase antibody inhibited Eap-induced platelet activation. The effect of Eap on platelets and protein disulfide isomerase activity was completely blocked by glycosaminoglycans. Inhibition by the hydrophobic probe bis(1-anilinonaphthalene 8-sulfonate) suggested the involvement of hydrophobic sites in protein disulfide isomerase and platelet activation by Eap., Conclusions: In the present study, we found an additional and yet unknown mechanism of platelet activation by a bacterial adhesin, involving stimulation of thiol isomerases. The thiol isomerase stimulatory and prothrombotic features of a microbial secreted protein are probably not restricted to S aureus and Eap. Because many microorganisms are coated with amyloidogenic proteins, it is likely that the observed mechanism is a more general one.

Objective: To explore the value of flow cytometry in anaphylactic shock diagnosis by CD63 expression being detected using flow cytometry to conform the activation of basophils., Methods: Sixteen rats were randomly divided into two groups: control group and anaphylactic shock group. The model of anaphylactic shock rat with ovalbumin injection was established. CD63, CD45 and CD203c antibody combination, flow cytometry was employed to detected blood basophil CD63 expression. Immunofluorescence method was employed to observe the CD63 immunofluorescence staining in the rat lung tissue., Results: (1) Pure basophils were obtained by CD45 and CD203c gating. (2) The percentages of basophils CD63 were (17.34 +/- 2.04)% and (1.52 +/- 0.35)% in the experimental and control group, respectively. The differences between two groups were statistically significant (P < 0.01). (3) Compared with the control group, the expression of CD63 in basophils increased in anaphylactic shock lung tissue., Conclusion: The detection of CD63 by flow cytometry could be the supplement of vivo allergic reactions and have good clinical value.

Background: Microparticles are known to be increased in various malignancies. In this prospective study, microparticle levels were evaluated in patients with benign and malignant ovarian lesions., Patients and Methods: Microparticles from platelets/megakaryocytes, activated platelets and endothelial cells, tissue factor exposing microparticles and D-dimer values were examined in patients with newly diagnosed ovarian lesions before surgery, and were correlated with tumor histology., Results: Higher counts of CD63-positive microparticles were detected in patients with ovarian cancer [mean=276×10(6) (range: 64-948)/l; n=12] as compared to patients with benign ovarian tumors [146×10(6) (45-390)/l; n=21; p=0.014]. D-dimer values were also increased in patients with cancer [860 (180-4500) ng/l versus 280 (170-2720) ng/l; p=0.001]., Conclusion: Elevated levels of CD63-positive microparticles and D-dimer reflect the procoagulant phenotype of these patients. However, for the discrimination between benign and malignant ovarian tumors, measuring preoperative levels of microparticles does not seem to be helpful.

Specific immunotherapy (SIT) of hymenoptera venom allergy, in particular, has become an example for the effectiveness of the treatment of IgE-mediated allergic diseases. In vitro diagnostic procedures and the measurement of serum tryptase for risk assessment are well-established in the process of diagnose finding and therapy preparation. For monitoring and validation of the effectiveness of the SIT, however, in vitro diagnostic procedures remain controversial. Potentially useful approaches include detection of specific IgE, specific IgG4, basophile activation - represented by the CD 63 expression - and the lymphocyte proliferation and its IL10 release. Preliminary data suggest that the latter method appear appropriate, whereas the detection of basophile activation did not produce definite results.