Sensing Acute Cellular Rejection in Liver Transplant Patients Using Liver-Derived Extracellular Particles: A Prospective, Observational Study.

Acute cellular rejection (ACR) after liver transplantation (LT) goes along with allograft dysfunction, which is diagnosed by liver biopsy and concomitant histological analysis, representing the gold standard in clinical practice. Yet, liver biopsies are invasive, costly, time-intensive and require expert knowledge. Herein we present substantial evidence that blood plasma residing peripheral liver-derived extracellular particles (EP) could be employed to diagnose ACR non-invasively. In vitro experiments showed organ-specific EP release from primary human hepatocytes under immunological stress. Secondly, analysis of consecutive LT patients (n=11) revealed significant heightened EP concentrations days before ACR. By conducting a diagnostic accuracy study (n = 69, DRKS00011631), we explored the viability of using EP as a liquid biopsy for diagnosing ACR following LT. Consequently, novel EP populations in samples were identified using visualization of t-distributed stochastic neighbor embedding (viSNE) and self-organizing maps (FlowSOM) algorithms. As a result, the ASGR1+CD130+Annexin V+ EP subpopulation exhibited the highest accuracy for predicting ACR (area under the curve: 0.80, 95% confidence interval [CI], 0.70-0.90), with diagnostic sensitivity and specificity of 100% (95% CI, 81.67-100.0%) and 68.5% (95% CI, 55.3-79.3%), respectively. In summary, this new EP subpopulation presented the highest diagnostic accuracy for detecting ACR in LT patients.

Frail older adults show a distinct plasma microvesicle profile suggesting a prothrombotic and proinflammatory phenotype.

In a global context of advanced aging, geriatric diseases such as frailty syndrome face challenges in the search for biomarkers and preventive strategies. Frailty has been associated with atherothrombotic pathologies. Circulating microvesicles (cMVs), phospholipid-rich vesicles with a size of 0.1-1.0 µm, have been shown to participate in atherothrombosis onset and progression. We have hypothesized that cMVs from platelets, and vascular and immune cells, are increased in frail older adults. To verify this, a prevalent-case control study was designed with 28 frail older and 27 nonfrail older adults older than 64 years. Frailty was defined by Fried's phenotype. Total cMVs, annexin V positive (AV+)-cMVs, and annexin V negative (AV- )-cMVs derived from blood and vascular cells were measured by flow cytometry. In the analysis of total cMVs, the frail group presented higher levels of CD14+ /CD142+ (p = .042), CD41a+ /CD142+ (p = .041), and CD56+ (p = .025), CD14+ cMVs (p = .043), and CD16+ /CD14+ (p = .019) cMVs levels. Within the phosphatidylserine-exposing cMVs (AV+ ), the frail group showed higher CD14+ /AV+ (p = .044), CD9+ /AV+ (p = .031), P2RY12+ /AV+ (p = .028), and CD235a+ /AV+ (p = .043) cMVs concentrations. Finally, within AV- cMVs, the frail group showed higher CD142+ /CD41a+ /AV- cMVs concentrations originated from platelets (p = .027), CD56+ /AV- originated from natural killer cells (p = .022), and CD34+ /AV- cMVs from hematopoietic stem cells (p = .037). In summary, frail older adults present higher concentrations of platelet-, leukocyte-, and hematopoietic cell-derived cMVs compared to robust age-matched older adults. These cMVs may be involved in the deregulation of the immune system, endothelial damage, and increased risk of thrombosis associated with frailty.

Annexin V positive microvesicles are elevated and correlate with flow rate in patients receiving veno-arterial extracorporeal membrane oxygenation.

OBJECTIVES: Veno-arterial extracorporeal membrane oxygenation (VA-ECMO) is used in critically ill patients requiring haemodynamic support. Microvesicles (MV) are released by activated blood cells acting as mediators of intercellular communication. We aimed to determine MV count and composition over time in patients with VA-ECMO and explore what drives MV formation. METHODS: VA-ECMO patients and healthy controls were recruited prospectively, and blood was taken at different time points (day 0, 1, 3 after ECMO placement and after explantation) for MV analysis. RESULTS: Annexin V positive MV were increased in patients (n = 14, mean age = 61.4 ± 9.0 years, 11 males, 3 females) compared to healthy controls (n = 6, Annexin V positive MV count per millilitre day 1 versus healthy controls: 2.3 × 106 vs 1.3 × 105, P < 0.001). Furthermore, patients had higher proportions of endothelial and leukocyte MV [leukocyte MV day 1 versus healthy controls (%): 32.8 vs 17.5, P = 0.001; endothelial MV day 1 versus healthy controls (%): 10.5 vs 5.5, P = 0.01]. Annexin V positive and leucocyte MV correlated with the flow rate (r = 0.46, P = 0.01). CONCLUSIONS: Patients on VA-ECMO have increased levels of circulating MV and a changed MV composition. Our data support the hypothesis that MV release may be driven by higher flow rate and cellular activation in the extracorporeal circuit leading to poor outcomes in these patients. CLINICAL TRIAL REGISTRATION NUMBER: German Clinical Trials Register-ID: DRKS00011106.

Anti-annexin V autoantibodies and vascular abnormalities in systemic sclerosis: a longitudinal study

Abstract Background: Annexins are a group of conserved proteins which exert several regulatory functions on various cellular activities. Increased frequency and levels of antibodies against annexin V have already been observed in several autoimmune diseases including systemic sclerosis (SSc), but their role as a vascular biomarker is unknown. The aim of this study was to determine the serum levels and the dynamical behavior of anti-annexin V antibodies over a 24 months follow-up in patients with SSc. Methods: In this bicentric cross-sectional study, 70 patients with SSc were consecutively selected from March 2016 to April 2017. Demographic and clinical features, including the presence of active DUs, were collected. Serum anti-annexin V IgG and IgM antibodies were measured at baseline and after 6, 12 and 24 months of follow-up. Videocapillaroscopy was performed in all patients. Results: Among the 70 SSc patients included anti-annexin V IgG was found in 11 patients (15.7%) (range of 15.88-39.48 U/mL) and anti-annexin V IgM in 10 patients (14.3%) (range of 14.16-22.69 U/mL) at baseline. During follow-up, the number of patients who were positive for anti-annexin V IgG and IgM remained stable over 24 months. Among the patients with positive anti-annexin V IgG at baseline the frequency of patients with necrosis or amputation of extremities, forced vital capacity less than 70% and pulmonary arterial hypertension (PAH) was significantly higher than in patients with negative anti-annexin V IgG antibodies. Patients with anti-annexin V IgG had also a higher Raynaud's Condition Score and a higher Health Assessment Questionnaire Disability Index (HAQ-DI) than patients without these antibodies at baseline. Patients with positive anti-annexin V IgM at baseline presented a higher frequency of PAH, compared to those with negative anti-annexin V IgM at baseline. Conclusions: Anti-annexin V antibodies are stable and do not change their positivity during a 24 month follow-up in SSc patients. Anti-annexin V IgG was associated with more severe interstitial lung involvement and digital microangiopathy, and patients with anti-annexin V IgG or IgM had a higher occurrence of PAH indicating an association of these biomarker with more severe disease.(AU)

Serum Annexin V and Anti-Annexin V levels and their relationship with metabolic parameters in patients with type 2 diabetes

SUMMARY BACKGROUND We investigated the serum annexin V and anti-annexin V levels and their relationship with metabolic parameters in patients recently diagnosed type 2 diabetic. METHODS A total of 143 patients recently diagnosed type 2 diabetes and 133 control subjects were included in the study. Body mass index (BMI), hs-CRP, HOMA-IR, carotid intima-media thickness, and serum levels of annexin V and anti-annexin V were investigated. RESULTS HOMA-IR, serum hs-CRP, and carotid intima-media thickness were found to be statistically significant. The Pearson correlation analysis revealed a statistically significant positive relationship between the carotid intima-media thickness and the annexin V level (r=0.29, p=0.006*). A statistically significant positive relationship was also detected between the Annexin V level and level of serum hs-CRP (r=0.29 p=0.006*). CONCLUSION A positive relationship was observed between the carotid intima-media thickness and annexin V at the end of our investigation. In this regard, we also believe that serum levels of annexin V may be increased for cardiovascular protection in the elevation of carotid intima-media thickness.

RESUMO OBJETIVO Investigar os níveis séricos de anexina V e antianexina V e sua relação com os parâmetros metabólicos em pacientes diabéticos tipo 2 recém-diagnosticados. MÉTODOS Foram incluídos no estudo 143 pacientes e 133 controles com diabetes tipo 2 recém-diagnosticado. O índice de massa corporal (IMC), PCR-as, Homa-IR, espessura íntima média carotídea e níveis séricos de anexina V e antianexina V foram investigados. RESULTADOS O Homa-IR, a PCR-s do soro e a espessura média da carótida foram estatisticamente significantes. A análise de correlação de Pearson revelou uma relação positiva estatisticamente significante entre a espessura média da carótida e anexina V (r=0,29; p=0,006 *). Foi também detectada uma relação positiva estatisticamente significativa entre o nível de anexina V e o nível sérico de PCR-as (r=0,29, p=0,006*). CONCLUSÃO Também foi observada uma relação positiva entre a espessura média da carótida e anexina V no final da nossa investigação. A esse respeito, também pensamos que os níveis séricos de anexina V podem ser aumentados para proteção cardiovascular na elevação da espessura média da carótida.

Altered signature of apoptotic endothelial cell-derived microvesicles predicts chronic heart failure phenotypes.

Aim: to evaluate the associations between signatures of apoptotic endothelial cell-derived microvesicles (MVs) with phenotypes of chronic heart failure (HF). Methods: The study cohort consisted of 388 prospectively involved subjects with HF patients with predominantly reduced left ventricular ejection fraction (HFrEF), HF with preserved ejection fraction (HFpEF) and HF with mid-range ejection fraction (HFmrEF). All biomarkers were measured at baseline. Results: The number of circulating CD31+/annexin V+ MVs in HFrEF and HFmrEF patients was similar. The number of circulating CD144+/annexin V+ MVs in HFrEF patients was significantly higher than HFmrEF and HFpEF. We determined that a combination of number of circulating CD31+/annexin V+ MVs and Gal-3 was the best predictor of HFpEF and that number of circulating CD144+/annexin V+ MVs is able to increase predictive capabilities of soluble ST2 (sST2) and Gal-3 for HFrEF. Conclusion: We found that the number of circulating CD31+/annexin V+ MVs may improve a predictive capacity for conventional HF biomarkers.

Role of red blood cells "annexin V" and platelets "P-selectin" in patients with thalassemia.

OBJECTIVE/BACKGROUND: Certain hemostatic anomalies found in patients with thalassemia suggest the existence of a chronic hypercoagulable state. Several etiologic factors may play a role in the pathogenesis of the hypercoagulable state in those patients. One of these factors is abnormal thalassemic red blood cells (RBCs), which may provide a procoagulant. To substantiate these findings, we measured the ability of RBCs from thalassemia patients to bind annexin V with increased fraction of platelets carrying the activation marker CD62P (P-selectin). To study the expression of RBC annexin V and platelets P-selectin in study patients (those with thalassemia major, thalassemia intermedia, thalassemia minor) and control group, four-color flow cytometry was performed and the correlation between these two markers was evaluated. METHODS: A case-control study was conducted on 50 ß-thalassemia patients (10 patients with thalassemia minor, 30 patients with thalassemia major, and 10 patients with thalassemia intermedia, with 10 normal adult volunteers as a control) from June 2016 to March 2017. Flow cytometry was used to study the expression of anionic phospholipids (Annexin V) on the RBCs and CD62P (P-selectin) on the activated platelet. RESULTS: The mean expression of annexin V in patients with thalassemia major and intermedia was significantly higher than that in the control group and patients with thalassemia minor. Although the mean expression was higher in patients with thalassemia intermedia than in those with thalassemia major, it was not statistically significant. CONCLUSION: The mean expression of platelets P-selectin in patients with thalassemia major and thalassemia intermedia was significantly higher than that in controls and patients with thalassemia minor. However, its expression was significantly higher in patients with thalassemia intermedia than in those with thalassemia major. Annexin V also showed a positive correlation with P-selectin, and both markers positively correlated with regularity of blood transfusion.

Apoptosis: A biomarker of high-risk phenotype in pediatric acute myeloid leukemia?

INTRODUCTION: Dysregulation of apoptosis has been explored in acute myeloid leukemia (AML); yet, its correlation with clinical outcomes in pediatric AML is unknown. This study was aimed to analyze percentage of apoptosis and apoptosis mediated through the intrinsic pathway with clinical outcomes in patients with pediatric AML. METHODS: This prospective study included pediatric AML patients enrolled from July 2013 to August 2016. Annexin-V (marker of total apoptosis) and caspase-9 expression (marker of intrinsic pathway) was determined in baseline bone marrow (BM) samples by flow cytometery and compared with controls (unaffected BM of solid tumors and peripheral blood [PB] of unaffected siblings). Overall survival (OS) and event-free survival (EFS) were compared using log-rank test. RESULTS: A total of 151 AML patients were enrolled, median age 10 (range: 0.7-18 years). Annexin-V expression in blast cells was significantly high in AML patients as compared to BM of subjects with solid tumors (P = 0.01) and PB of healthy subjects (P = 0.04). Caspase-9 expression in blast cells was not significantly different. Median annexin-V expression was significantly higher in patients with WBC count ≥11 000/mm3 (P = 0.02), poor-risk cytogenetics (P = 0.02), the absence of RUNX1-RUNX1T1 translocation (P = 0.004), and the absence of NPM1 mutation (P = 0.05). Patients with high annexin-V expression had significantly inferior OS (P = 0.05) in univariate analysis but not in multivariate analysis (P = 0.32). CONCLUSION: Apoptosis as a whole was found to be activated in baseline BM samples of AML patients. High apoptosis may be associated with high-risk phenotype in this disease.

Validation of a Gravitational Model to Study Local Endogenous Biomarkers in Chronic Venous Insufficiency.

OBJECTIVE/BACKGROUND: Unlike most systemic chronic diseases, chronic venous insufficiency (CVI) is ideal to study using endogenous biomarkers. The stimulus causing damage can be turned on and off with gravitational positioning and venous blood samples can be taken locally. Annexin V (apoptosis) and microparticles (cell membrane debris) were used as markers of cell destruction, with matrix metalloproteinases (MMPs) as markers of tissue remodelling. The aim of this proof of concept study was to validate a gravitational model by investigating whether standing induced biochemical stress and whether recovery occurs on lying and after compression. METHODS: Fourteen patients (C4a-b) and 14 volunteers (C0-1) were tested under three supervised laboratory conditions for 1 h on separate days: (i) stationary standing on a small paper square; (ii) lying with both legs elevated 20°; (iii) compression standing using a 23-32 mmHg below knee stocking. Immediately after each condition, venous blood was withdrawn from the ankle. Commercial enzyme linked immunosorbent assay kits were used for batch analysis of the plasma samples. RESULTS: Median (interquartile range [IQR]) values of annexin V (AU/mL) and microparticles (nM) standing were as follows: volunteers 2.9 (2 - 3.4) and 10.2 (8.8 - 13.8), and patients 2.2 (1.3 - 6) and 11.3 (7.7 - 20), respectively. Significant reductions were observed lying: volunteers 2.1 (1.5 - 2.7; p = .019) and 8.5 (7.4 - 9.4; p = .041), patients 1.7 (1.2 - 2.7; p = .004) and 8.5 (7.0 - 11.4; p = .041), respectively. Globally, all median MMP values in the patients reduced with lying and with compression versus standing (p = .004). Individually, significant reductions occurred in MMPs 2 and 13 with compression and MMPs 3, 7, 8, 9, 10, and 12 on lying. Lying was more effective at reducing MMP levels than compression. CONCLUSION: Annexin V and microparticle concentrations are responsive to elevation and compression after 1 h. In the patients, all the tested MMPs decreased after lying and with compression versus standing. This model provides evidence supporting gravitational protection in the treatment of CVI.

A method for red blood cell biotinylation in a closed system.

BACKGROUND: Several circumstances require the accurate measurement of red blood cell (RBC) survival and clearance, such as determination of posttransfusion recovery of stored RBCs to investigate the effect of new additive solutions. To this end, biotin as a marker of RBCs to track donor RBCs in the blood of the recipient has been used in many studies. However, so far only experimental, nonvalidated, biotin-labeled red cell concentrates (RCCs) are transfused. The goal of this study was to produce a standardized biotin-labeled RCC product in a fast, simple, and sterile manner that can be used for clinical research and for the evaluation of new blood products according to Good Practice Guidelines (GPG) for blood establishments. STUDY DESIGN AND METHODS: RCC fractions were labeled with two different concentrations of biotinylation reagent in a closed system, to prevent bacterial contamination of the end product. Using flow cytometry, the reproducibility and robustness of the biotin labeling was assessed, as well as the stability of the biotin label on the (un-)irradiated RCC fraction. Additionally, parameters such as phosphatidylserine (PS) exposure, sodium (Na), potassium (K), free hemoglobin, adenosine triphosphate (ATP), pH, and morphology were determined prior to and after biotin labeling to rule out detrimental effects of the labeling procedure on the RCC. RESULTS: Our data show that RCCs can be labeled under sterile conditions in a closed system with two different biotinylation reagent concentrations, without affecting the biological activity. CONCLUSION: An easy, rapid (<2 hr), and robust method was developed to manufacture biotin-labeled RCCs for clinical research compliant to GPG.

Platelet-derived microparticles regulates thrombin generation via phophatidylserine in abdominal sepsis.

Sepsis is associated with dysfunctional coagulation. Recent data suggest that platelets play a role in sepsis by promoting neutrophil accumulation. Herein, we show that cecal ligation and puncture (CLP) triggered systemic inflammation, which is characterized by formation of IL-6 and CXC chemokines as well as neutrophil accumulation in the lung. Platelet depletion decreased neutrophil accumulation, IL-6, and CXC chemokines formation in septic lungs. Depletion of platelets increased peak thrombin formation and total thrombin generation (TG) in plasma from septic animals. CLP elevated circulating levels of platelet-derived microparticles (PMPs). In vitro generated PMPs were a potent inducer of TG. Interestingly, in vitro wild-type recombinant annexin V abolished PMP-induced thrombin formation whereas a mutant annexin V protein, which does not bind to phosphatidylserine (PS), had no effect. Administration of wild-type, but not mutant annexin V, significantly inhibited thrombin formation in septic animals. Moreover, CLP-induced formation of thrombin-antithrombin complexes were reduced in platelet-depleted mice and in animals pretreated with annexin V. PMP-induced TG attenuated in FXII- and FVII-deficient plasma. These findings suggest that sepsis-induced TG is dependent on platelets. Moreover, PMPs formed in sepsis are a potent inducer of TG via PS exposure, and activation of both the intrinsic and extrinsic pathway of coagulation. In conclusion, these observations suggest that PMPs and PS play an important role in dysfunctional coagulation in abdominal sepsis.

Expression of annexin A5 in serum and tumor tissue of patients with colon cancer and its clinical significance.

AIM: To investigate the expression of annexin A5 in serum and tumor tissue of patients with colon cancer and to analyze its clinical significance. METHODS: Ninety-three patients with colon cancer treated at our hospital between February 2013 and March 2016 were included in an observation group, and 40 healthy individuals were included in a control group. Enzyme-linked immunosorbent assay was performed to determine the serum level of annexin A5, while immunohistochemistry was performed to determine the expression of annexin A5 in cancer tissues. RESULTS: The serum level of annexin A5 was 0.184 ± 0.043 ng/mL in the observation group, which was significantly higher than that in the control group (P < 0.05). Annexin A5 expression was detected in 79.31% of the patients with lymph node metastasis, which was significantly higher than that in patients without lymph node metastasis (P < 0.05). Moreover, annexin A5 expression was detected in 86.96% of the patients with stage III to IV disease, which was significantly higher than that in patients with stage I to II disease (P < 0.05). The serum level of annexin A5 was 0.215 ± 0.044 ng/mL in patients whose tumors were positive for annexin A5 expression, which was significantly higher than that in patients whose tumors were negative for annexin A5 expression (P < 0.05). The serum level of annexin A5 was correlated with annexin A5 expression in colon cancer tissues (r = 0.312, P < 0.05). When a cutoff value of > 0.148 ng/mL for serum level of annexin A5 was used in the diagnosis of colon cancer, the sensitivity was 83.90%, and the specificity was 57.50%. CONCLUSION: For patients with colon cancer, annexin A5 expression in cancer tissues is related to lymph node metastasis and tumor grade. Serum level of annexin A5 is related to annexin A5 expression in cancer tissues and is of diagnostic relevance.

Normalized levels of red blood cells expressing phosphatidylserine, their microparticles, and activated platelets in young patients with ß-thalassemia following bone marrow transplantation.

Bone marrow transplantation (BMT) serves as the only curative treatment for patients with ß-thalassemia major; however, hemostatic changes have been observed in these BMT patients. Aggregability of thalassemic red blood cells (RBCs) and increased red blood cell-derived microparticles (RMPs) expressing phosphatidylserine (PS) are thought to participate in thromboembolic events by initially triggering platelet activation. To our knowledge, there has been no report providing quantitation of these circulating PS-expressing RBCs and RMPs in young ß-thalassemia patients after BMT. Whole blood from each subject was fluorescently labeled to detect RBC markers (CD235a) and annexin-V together with the known number TruCount™ beads. PS-expressing RBCs, RMPs, and activated platelets were identified by flow cytometry. In our randomized study, we found the decreased levels of three aforementioned factors compared to levels in patients receiving regular blood transfusion (RT). This study showed that BMT in ß-thalassemia patients decreases the levels of circulating PS-expressing RBCs, their MPs, and procoagulant platelets when compared to patients who received RT. Normalized levels of these coagulation markers may provide the supportive evidence of the effectiveness of BMT for curing thalassemia.

Bleeding tendency and platelet function during treatment with romiplostim in children with severe immune thrombocytopenic purpura.

It has been suggested that platelet function in chronic immune thrombocytopenic purpura (ITP) may be abnormal. Thrombopoietin mimetics used for treatment can affect it, but the data remain limited. We investigated platelet function of 20 children diagnosed with severe ITP (aged 1-16 years, 12 females and eight males). Platelet functional activity in whole blood was characterized by flow cytometry before and after stimulation with SFLLRN plus collagen-related peptide. Levels of CD42b, PAC1, and CD62P, but not CD61 or annexin V, were significantly increased (P < 0.05) in resting platelets of patients before treatment compared with healthy donors. On average, PAC1 and CD62P in patients after activation were also significantly elevated, although some patients failed to activate integrins. Romiplostim (1-15 µg/kg/week s.c.) was prescribed to seven patients, with clinical improvement in six. Interestingly, one patient had clinical improvement without platelet count increase. Eltrombopag (25-75 mg/day p.o.) was given to four patients, with positive response in one. Others switched to romiplostim, with one stable positive response, one unstable positive response, and one non-responding. Platelet quality improved with romiplostim treatment, and their parameters approached the normal values. Our results suggest that platelets in children with severe ITP are pre-activated and abnormal, but improve with treatment.

Cancer-associated circulating large extracellular vesicles in cholangiocarcinoma and hepatocellular carcinoma.

BACKGROUND & AIMS: Large extracellular vesicles, specifically AnnexinV+ EpCAM+ CD147+ tumour-associated microparticles (taMPs), facilitate the detection of colorectal carcinoma (CRC), non-small cell lung carcinoma (NSCLC) as well as pancreas carcinoma (PaCa). Here we assess the diagnostic value of taMPs for detection and monitoring of hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA). Specifically, the aim of this study was to differentiate liver taMPs from other cancer taMPs, such as CRC and NSCLC. METHODS: Fluorescence-activated cell scanning (FACS) was applied to detect various taMP populations in patients' sera that were associated with the presence of a tumour (AnnexinV+ EpCAM+ CD147+ taMPs) or could discriminate between cirrhosis (due to HCV or HBV) and liver cancers (AnnexinV+ EpCAM+ ASGPR1+ taMPs). In total 172 patients with liver cancer (HCC or CCA), 54 with cirrhosis and no liver neoplasia, and 202 control subjects were enrolled. RESULTS: The results indicate that AnnexinV+ EpCAM+ CD147+ taMPs were elevated in HCC and CCA. Furthermore, AnnexinV+ EpCAM+ ASGPR1+ CD133+ taMPs allowed the distinction of liver malignancies (HCC or CCA) and cirrhosis from tumour-free individuals and, more importantly, from patients carrying other non-liver cancers. In addition, AnnexinV+ EpCAM+ ASGPR1+ taMPs were increased in liver cancer-bearing patients compared to patients with cirrhosis that lacked any detectable liver malignancy. The smallest sizes of successfully detected cancers were ranging between 11-15mm. AnnexinV+ EpCAM+ ASGPR1+ taMPs decreased at 7days after curative R0 tumour resection suggesting close correlations with tumour presence. ROC values, sensitivity/specificity scores and positive/negative predictive values (>78%) indicated a potent diagnostic accuracy of AnnexinV+ EpCAM+ ASGPR1+ taMPs. CONCLUSION: These data provide strong evidence that AnnexinV+ EpCAM+ ASGPR1+ taMPs are a novel biomarker of HCC and CCA liquid biopsy that permit a non-invasive assessment of the presence and possible extent of these cancers in patients with advanced liver diseases. LAY SUMMARY: Microparticles (MPs) are small vesicles that bleb from the membrane of every cell, including cancer cells, and are released to circulate in the bloodstream. Since their surface composition is similar to the surface of their underlying parental cell, MPs from the bloodstream can be isolated and by screening their surface components, the presence of their parental cells can be identified. This way, it was possible to detect and discriminate between patients bearing liver cancer and chronic liver cirrhosis.

[Biochemical and immunological markers of autoimmune thyroiditis]. / Biokhimicheskie i immunologicheskie markery khronicheskogo limfotsitarnogo tireoidita.

Correlations between biochemical and immunological markers of programmed cell death (apoptosis), and the functional state of the thyroid gland (hyperthyroidism, euthyroidism, hypothyroidism) have been investigated in autoimmune thyroiditis (AT) (also known as chronic autoimmune thyroiditis). Annexin V, TRAIL and TNF-a, as well as DNA-hydrolyzing antibodies were used as the main markers. Increased levels of TRAIL were found in the serum of AT patients (hyperthyroidism>hypothyroidism>euthyroidism) compared with healthy individuals. The highest frequency of antibodies to denatured DNA (Abs-dDNA) had the highest frequency in AT patients (97%) compared with healthy controls. Among these patients, 75% had hyperthyroidism, 85% had hypothyroidism, and 84.7% had euthyroidism. Abs hydrolyzing activity demonstrated correlation dependence with symptoms of the thyroid dysfunction.

Annexin A5 promotes macrophage activation and contributes to pulmonary fibrosis induced by silica particles.

OBJECTIVE: To investigate the contributions and underlying molecular mechanisms of annexin A5 toward silica-induced pulmonary fibrosis. METHODS: Male C57BL/6 mice were randomly divided into three groups and instilled intratracheally with silica, saline, or air. Mice were euthanized at 3, 7, 14, or 28 days following treatment. Annexin A5 levels in serum and lung tissues were detected by enzyme-linked immunosorbant assay (ELISA) assays or Western blots. The association of annexin A5 levels with silica-induced lung fibrosis was further investigated in the macrophage cell line, RAW264.7. Following exposure of these cells to silica at a concentration of 200 µg/ml for 6 or 12 h, the expression levels of transforming growth factor ß1 (TGF-ß1), interleukin 1α (IL-1α), Fas ligand (FasL), and their downstream targets were evaluated by Western blots. Furthermore, annexin A5 and FasL were knocked down by small interfering ribonucleic acid (siRNA) and TGF-ß1 secretion into the cell culture medium was measured by ELISA assays or Western blots. RESULTS: Mice treated with silica demonstrated lung fibrosis at 28 days following exposure, whereas, in controls, only mild and transient inflammation was evident at day 3 and day 7 postinstillation and was not present at day 14. Furthermore, silica-exposed mice exhibited significantly (p < 0.05) elevated levels of annexin A5 in serum and lung tissues, relative to control groups. Consistent with these findings, silica exposure of RAW264.7 cells for 6 or 12 h, led to an annexin A5-dependent increase in the expression levels of TGF-ß1, IL-1α, FasL, and their downstream target molecules. These silica-induced changes were reversed by siRNA-mediated knockdown of annexin A5, but downregulation of FasL led to increased annexin A5 expression and reduced levels of TGF-ß1, IL-1α, and FasL downstream target molecules. CONCLUSIONS: These findings define a role of annexin A5 in promoting macrophage activation via Fas/FasL pathways in silica-induced lung fibrosis.

Annexin A5 haplotypes in familial hypercholesterolemia: lack of association with carotid intima-media thickness and cardiovascular disease risk.

OBJECTIVE: Annexin A5 (ANXA5) has been suggested to possess antiatherogenic properties. We investigated whether ANXA5 genetic variations and plasma ANXA5 levels were associated with carotid atherosclerosis and contributed to cardiovascular disease (CVD) risk in patients with familial hypercholesterolemia (FH). METHODS: We sequenced the promoter region and exon 2 of ANXA5 in 284 FH patients from the ASAP (Atorvastatin versus Simvastatin on Atherosclerosis Progression) trial. Common haplotypes (H) were constructed based on seven single nucleotide polymorphisms (SNPs). We studied whether plasma ANXA5 levels or ANXA5 haplotypes were associated with the extent of atherosclerosis (evaluated by carotid intima-media thickness (IMT). The association between ANXA5 haplotypes and the risk for CVD events was investigated in 1730 FH patients from the GIRaFH (Genetic Identification of Risk factors in Familial Hypercholesterolemia) study. RESULTS: In ASAP, individuals carrying the ANXA5 haplotype H2 exhibited lower plasma ANXA5 levels, whereas H4 carriers had increased levels of circulating ANXA5 compared to non-carriers. Plasma ANXA5 levels were not associated with carotid IMT. None of the four ANXA5 haplotypes correlated with the age-related IMT progression (ASAP study) or contributed to CVD risk (GIRaFH cohort). CONCLUSIONS: Both ANXA5 haplotypes and plasma ANXA5 levels were not associated with carotid IMT progression or CVD risk in FH patients.

Young women with polycystic ovary syndrome have raised levels of circulating annexin V-positive platelet microparticles.

STUDY QUESTION: Are circulating microparticles (MPs) altered in young women with polycystic ovary syndrome (PCOS)? SUMMARY ANSWER: Women with PCOS have elevated concentrations of circulating platelet-derived MPs, which exhibit increased annexin V binding and altered microRNA (miR) profiles compared with healthy volunteers. WHAT IS KNOWN ALREADY: Some studies have shown that cardiovascular risk is increased in young women with PCOS but the mechanisms by which this occurs are uncertain. Circulating MPs are elevated in patients with cardiovascular disease but the characteristics of MPs in patients with PCOS are unclear. STUDY DESIGN, SIZE, DURATION: Case-control study comprising 17 women with PCOS (mean ± SD; age 31 ± 7 years, BMI 29 ± 6 kg/m(2)) and 18 healthy volunteers (age 31 ± 6 years, BMI 30 ± 6 kg/m(2)). PARTICIPANTS/MATERIALS, SETTING, METHODS: The study was conducted in a University hospital. Nanoparticle tracking analysis (NTA) and flow cytometry (CD41 platelet, CD11b monocyte, CD144 endothelial) were used to determine MP size, concentration, cellular origin and annexin V positivity (reflecting phosphatidylserine exposure). Fatty acid analysis was performed by gas chromatography and MP miR expression profiles were compared by microarray. MAIN RESULTS AND THE ROLE OF CHANCE: PCOS subjects showed increased MP concentrations compared with healthy volunteers (mean ± SD; 11.5 ± 5 × 10(12)/ml versus 10.0 ± 4 × 10(12)/ml, respectively; P = 0.03), which correlated with the homeostasis model of insulin resistance (r = 0.53, P = 0.03). This difference was predominantly seen in MPs whose size was in the small exosomal range (<150 nm in diameter, P< 0.05). PCOS patients showed a greater percentage of annexin V(+) MPs compared with healthy volunteers (84 ± 18 versus 74 ± 24%, respectively, P = 0.05) but the cellular origin of MPs, which were predominantly platelet-derived (PCOS: 99 ± 0.9%; controls: 99 ± 2.5%), did not differ. MP fatty acid concentration and composition was similar between groups but 16 miRs were differentially expressed (P < 0.05). LIMITATIONS, REASON FOR CAUTION: Patients with PCOS were classified by the Rotterdam criteria, which describes a less severe metabolic phenotype than other definitions of the syndrome. Our findings may thus not be generalizable to all patients with PCOS. MicroRNA expression analysis was only undertaken in an exploratory subset of the overall study population hence, validation of our findings in a larger cohort is mandatory. Furthermore, miR levels were unaltered for the highly expressed miRs and it is unclear whether differences in the lowly expressed miRs carries pathological relevance. WIDER IMPLICATIONS OF THE FINDINGS: This study suggests that women with PCOS have an altered MP profile but further studies are needed to confirm this, to explore the mechanisms by which these alterations develop and to establish whether therapies that improve insulin sensitivity are able to reduce circulating MP concentrations. STUDY FUNDING/COMPETING INTERESTS: The study was funded by grants from the Wales Heart Research Institute and Mrs John Nixon Scholarship. The authors have no conflicts of interest to declare.

[The annexin 5 in serums of pregnant women and patients with particular types of cancer].

The detection of biomarkers to be applied to reveal different types of tumors is an actual demand of today. Since course of this disease can be asymptomatic these kinds of markers can be applied in widely-distributed screening procedures. Many types of biomarkers for detection of cancer exist. However all these markers are specific only for particular type of tumor and have no use in screening procedures for detection of this pathology. The Annexin 5, a Ca-depended phospholipid binding protein, was discovered in serums of all pregnant women on various stages of pregnancy. The study was implemented using method of latex agglutination. More than that, this protein was detected in serums of patients with several types of cancer. The possibility to apply annexin 5 as marker for screening of various types of cancer is considered.