Preliminary Study of Plasma Exosomal Tau as a Potential Biomarker for Chronic Traumatic Encephalopathy.

BACKGROUND: Chronic traumatic encephalopathy (CTE) is a tauopathy associated with prior exposure to repetitive head impacts, such as those incurred through American football and other collision sports. Diagnosis is made through neuropathological examination. Many of the clinical features of CTE are common in the general population, with and without a history of head impact exposure, making clinical diagnosis difficult. As is now common in the diagnosis of other neurodegenerative disorders, such as Alzheimer's disease, there is a need for methods to diagnose CTE during life through objective biomarkers. OBJECTIVE: The aim of this study was to examine tau-positive exosomes in plasma as a potential CTE biomarker. METHODS: Subjects were 78 former National Football League (NFL) players and 16 controls. Extracellular vesicles were isolated from plasma. Fluorescent nanoparticle tracking analysis was used to determine the number of vesicles staining positive for tau. RESULTS: The NFL group had higher exosomal tau than the control group (p <  0.0001). Exosomal tau discriminated between the groups, with 82% sensitivity, 100% specificity, 100% positive predictive value, and 53% negative predictive value. Within the NFL group, higher exosomal tau was associated with worse performance on tests of memory (p = 0.0126) and psychomotor speed (p = 0.0093). CONCLUSION: These preliminary findings suggest that exosomal tau in plasma may be an accurate, noninvasive CTE biomarker.

Exosome platform for diagnosis and monitoring of traumatic brain injury.

We have previously demonstrated the release of membranous structures by cells into their extracellular environment, which are termed exosomes, microvesicles or extracellular vesicles depending on specific characteristics, including size, composition and biogenesis pathway. With activation, injury, stress, transformation or infection, cells express proteins and RNAs associated with the cellular responses to these events. The exosomes released by these cells can exhibit an array of proteins, lipids and nucleic acids linked to these physiologic events. This review focuses on exosomes associated with traumatic brain injury, which may be both diagnostic and a causative factor in the progression of the injury. Based on current data, exosomes play essential roles as conveyers of intercellular communication and mediators of many of the pathological conditions associated with development, progression and therapeutic failures and cellular stress in a variety of pathologic conditions. These extracellular vesicles express components responsible for angiogenesis promotion, stromal remodelling, signal pathway activation through growth factor/receptor transfer, chemoresistance, immunologic activation and genetic exchange. These circulating exosomes not only represent a central mediator of the pro-inflammatory microenvironment linked with secondary brain injury, but their presence in the peripheral circulation may serve as a surrogate for biopsies, enabling real-time diagnosis and monitoring of neurodegenerative progression.

The origin, function, and diagnostic potential of RNA within extracellular vesicles present in human biological fluids.

We have previously demonstrated that tumor cells release membranous structures into their extracellular environment, which are termed exosomes, microvesicles or extracellular vesicles depending on specific characteristics, including size, composition and biogenesis pathway. These cell-derived vesicles can exhibit an array of proteins, lipids and nucleic acids derived from the originating tumor. This review focuses of the transcriptome (RNA) of these extracellular vesicles. Based on current data, these vesicular components play essential roles as conveyers of intercellular communication and mediators of many of the pathological conditions associated with cancer development, progression and therapeutic failures. These extracellular vesicles express components responsible for angiogenesis promotion, stromal remodeling, signal pathway activation through growth factor/receptor transfer, chemoresistance, and genetic exchange. These tumor-derived extracellular vesicles not only to represent a central mediator of the tumor microenvironment, but their presence in the peripheral circulation may serve as a surrogate for tumor biopsies, enabling real-time diagnosis and disease monitoring.

Alterations in antibody subclass immune reactivity to trophoblast-derived fetal fibronectin and α2-macroglobulin in women with recurrent pregnancy loss.

PROBLEM: Increasing evidence supports the involvement of complex antibody-mediated immunologic events at the decidua-trophoblast interface. Our objective is to define the humoral immune responses of pregnant women with a history of recurrent pregnancy loss (RPL) compared with gestation-age-matched and non-pregnant controls in terms of trophoblast-derived antigenic targets and IgG subclasses. METHOD OF STUDY: Immunoprecipitation and Western immunoblotting were performed to characterize IgG subclass reactivity to Sw.71 trophoblast-derived fetal fibronectin and alpha-2-macroglobulin, using serum obtained from first-trimester pregnant RPL subjects, gestation-age-matched controls, and non-pregnant controls. RESULTS: Using a generalized linear model, sera from women with a history of RPL exhibited increased IgG(3) immunoreactivity to trophoblast-derived fetal fibronectin and alpha-2-macroglobulin compared with controls (P < 0.001 and P < 0.001, respectively). CONCLUSION: IgG(3) reactivity in women with RPL may play a significant role in aberrant immune-regulatory mechanisms in early pregnancy. Further investigations into the role of autoantibodies against trophoblast-derived proteins in implantation and pregnancy are warranted.

Nanoparticle analysis of circulating cell-derived vesicles in ovarian cancer patients.

Cell-derived vesicles are recognized as essential components of intercellular communication, and many disease processes are associated with their aberrant composition and release. Circulating tumor-derived vesicles have major potential as biomarkers; however, the diagnostic use of exosomes is limited by the technology available for their objective characterization and measurement. In this study, we compare nanoparticle tracking analysis (NTA) with submicron particle analysis (SPA), dynamic light scattering (DLS), and electron microscopy (EM) to objectively define size distribution, number, and phenotype of circulating cell-derived vesicles from ovarian cancer patients. Using the NanoSight LM10 instrument, cell-derived vesicles were visualized by laser light scattering and analyzing Brownian motion of these vesicles captured by video. The NTA software calculates the size and total concentration of the vesicles in solution. Using vesicles isolated from ovarian cancer patients, we demonstrate that NTA can measure the size distributions of cell-derived vesicles comparable to other analysis instrumentation. Size determinations by NTA, SPA, and DLS were more objective and complete than that obtained with the commonly used EM approach. NTA can also define the total vesicle concentration. Furthermore, the use of fluorescent-labeled antibodies against specific markers with NTA allows the determination of the "phenotype" of the cell-derived vesicles.

Exosomes/microvesicles: mediators of cancer-associated immunosuppressive microenvironments.

Cancer cells, both in vivo and in vitro, have been demonstrated to release membranous structures, defined as microvesicles or exosomes, consisting of an array of macromolecules derived from the originating cells, including proteins, lipids, and nucleic acids. While only recently have the roles of these vesicular components in intercellular communication become elucidated, significant evidence has demonstrated that tumor exosomes can exert a broad array of detrimental effects on the immune system-ranging from apoptosis of activated cytotoxic T cells to impairment of monocyte differentiation into dendritic cells, to induction of myeloid-suppressive cells and T regulatory cells. Immunosuppressive exosomes of tumor origin can be found within neoplastic lesions and in biologic fluids from cancer patients, implying a potential role of these pathways in in vivo tumor progression and systemic paraneoplastic syndromes. Through the expression of molecules involved in angiogenesis promotion, stromal remodeling, signaling pathway activation through growth factor/receptor transfer, chemoresistance, and genetic intercellular exchange, tumor exosomes could represent a central mediator of the tumor microenvironment. By understanding the nature of these tumor-derived exosomes/microvesicles and their roles in mediating cancer progression and modulating the host immune response will significantly impact therapeutic approaches targeting exosomes.

Exosome isolation for proteomic analyses and RNA profiling.

While the existence of exosomes has been known for over three decades, they have garnered recent interest due to their potential diagnostic and therapeutic relevance. The expression and release of specific tumor-derived proteins into the peripheral circulation has served as the centerpiece of cancer screening and diagnosis. Recently, tissue-associated microRNA (miRNA) has been shown to be characteristic of tumor type and developmental origin, as well as exhibit diagnostic potential. Tumors actively release exosomes, exhibiting proteins and RNAs derived from the originating cell, into the peripheral circulation and other biologic fluids. Recently, we have demonstrated the presence of miRNAs within the RNA fraction of circulating tumor-derived exosomes. Currently, in over 75 investigations compiled in ExoCarta, over 2,300 proteins and 270 miRNAs have been linked with exosomes derived from biologic fluids. Our previous work has indicated that these circulating exosomal proteins and miRNAs can serve as surrogates for the tumor cell-associated counterparts, extending their diagnostic potential to asymptomatic individuals. In this chapter, we compare currently utilized methods for purifying exosomes for postisolation analyses. The exosomes derived from these approaches were assessed for quantity and quality of specific RNA populations and specific marker proteins. These results suggest that, while each method purifies exosomal material, circulating exosomes isolated by ExoQuick precipitation produces exosomal RNA and protein with greater purity and quantity than chromatography, ultracentrifugation, and DynaBeads. While this precipitation approach isolates exosomes in general and does not exhibit specificity for the originating cell, the increased quantity and quality of exosomal proteins and RNA should enhance the sensitivity and accuracy of down-stream analyses, such as qRT-PCR profiling of miRNA and mass spectrometric and electrophoretic analyses of exosomal proteins.

Human trophoblast-derived exosomal fibronectin induces pro-inflammatory IL-1ß production by macrophages.

PROBLEM Our previous studies demonstrated that trophoblast-derived exosomes induced synthesis and release of pro-inflammatory cytokines, including interleukin-1ß (IL-1ß) by macrophages. The objective of this study was to characterize the mechanism and receptors associated with this induction. METHOD OF STUDY Exosomes were isolated from Sw71 trophoblast-conditioned media by ultrafiltration and ultracentrifugation. Using macrophages isolated from normal donors, cytochalasin D was used to block exosome uptake. Induction of IL-1ß mRNA was investigated by qRT-PCR, pro-IL-1ß protein by western immunoblotting, and mature IL-1ß release by ELISA. RGD peptides were used to block fibronectin binding by macrophage α5ß1 integrin. RESULTS Uptake of exosomes by macrophages was completely blocked by pre-treatment with cytochalasin D. Although induction of some cytokines (such as C4A and CCL11) requires uptake, induction of IL-1ß occurred without exosome internalization. Cytochalasin D treatment did not inhibit exosome-mediated induction of IL-1ß mRNA, production of the pro-protein, or release of mature IL-1ß. Blocking of fibronectin binding using RGD peptides demonstrated the abrogation of exosome-mediated IL-1ß production. CONCLUSION Although trophoblast-derived exosomes have been demonstrated to induce IL-1ß, this is the first demonstration of IL-1ß induction by exosome-associated fibronectin. Based on this pro-inflammatory role of exosome-associated fibronectin, it may represent an important general immunoregulatory mechanism.

Morphologic and proteomic characterization of exosomes released by cultured extravillous trophoblast cells.

Exosomes represent an important intercellular communication vehicle, mediating events essential for the decidual microenvironment. While we have demonstrated exosome induction of pro-inflammatory cytokines, to date, no extensive characterization of trophoblast-derived exosomes has been provided. Our objective was to provide a morphologic and proteomic characterization of these exosomes. Exosomes were isolated from the conditioned media of Swan71 human trophoblast cells by ultrafiltration and ultracentrifugation. These were analyzed for density (sucrose density gradient centrifugation), morphology (electron microscopy), size (dynamic light scattering) and protein composition (Ion Trap mass spectrometry and western immunoblotting). Based on density gradient centrifugation, microvesicles from Sw71 cells exhibit a density between 1.134 and 1.173g/ml. Electron microscopy demonstrated that microvesicles from Sw71 cells exhibit the characteristic cup-shaped morphology of exosomes. Dynamic light scattering showed a bell-shaped curve, indicating a homogeneous population with a mean size of 165nm ± 0.5nm. Ion Trap mass spectrometry demonstrated the presence of exosome marker proteins (including CD81, Alix, cytoskeleton related proteins, and Rab family). The MS results were confirmed by western immunoblotting. Based on morphology, density, size and protein composition, we defined the release of exosomes from extravillous trophoblast cells and provide their first extensive characterization. This characterization is essential in furthering our understanding of "normal" early pregnancy.

Trophoblast-derived exosomes mediate monocyte recruitment and differentiation.

INTRODUCTION: trophoblast cells have been demonstrated to regulate monocyte migration and differentiation, leading to pro-inflammatory profiles. Because trophoblast cells release exosomes with immunoregulatory properties, trophoblast-derived exosomes are proposed to 'educate' monocytes, creating a pro-inflammatory environment. METHOD OF STUDY: exosomes were isolated from conditioned media of Swan71 cells by ultrafiltration and ultracentrifugation. Exosome-induced migration was assessed using a two-chamber system. Cytokine profiles were defined using cytokine arrays, and mRNA levels of affected cytokines were examined by qRT-PCR and ELISA. RESULTS: within 20 min, 8-10% of monocytes took up labeled exosomes isolated from Swan71 cells. Trophoblast-derived exosomes increased monocyte migration in a dose-dependent manner and produced significant increases in production of interleukin (IL)-1ß, IL-6, Serpin-E1, granulocyte colony-stimulating factor, granulocyte/monocyte colony-stimulating factor, and tumor necrosis factor-α. CONCLUSION: this study presents the initial demonstration that trophoblast-derived exosomes are capable of recruiting and 'educating' monocytes to produce pro-inflammatory cytokine/chemokine profiles in a cell-contact-independent manner.

Characterization of humoral responses of ovarian cancer patients: antibody subclasses and antigenic components.

OBJECTIVES: Current antigen-based diagnostic assays for ovarian cancers rely on intravasation of specific aberrantly expressed proteins and their achieving detectable steady-state concentrations, resulting in their inability to truly detect small early lesions. In contrast, tumor antigen immunorecognition is observed following initial transformation events. Our objective was to characterize humoral antitumor responses in terms of IgG subclasses generated and tumor antigens recognized. METHODS: For patients with benign and malignant ovarian disease, tumor-reactive IgG subclasses were characterized by Western immunoblotting. Antigen recognition patterns were analyzed by 2-dimensional electrophoresis and proteins exhibiting shared or stage-specific recognition were defined by mass spectrometry (MS) sequencing. RESULTS: Sera from ovarian cancer patients exhibited significantly greater immunoreactivities than either controls or women with benign disease. While late-stage patients recognized more proteins at greater intensity, stage-specific differential recognition patterns were observed in the IgG subclasses, with the greatest recognition appearing in IgG2 subclasses. Immunoreactivity in IgG2 and IgG3 from stage I and II patients appears to be most intense with nuclear antigens >40 kDa, while, in stage III patients, additional immunoreactivity was present in the <40 kDa components. Stage III patients also exhibited similar reaction with membrane antigens <40 kDa. Two-dimensional electrophoresis revealed 32 stage-linked antigenic differences with 11 in early-stage and 21 in late-stage ovarian cancer. CONCLUSIONS: Owing to the timing and stability of humoral responses, quantitation of IgG subclasses recognizing specific tumor antigens provides superior biomarkers for early cancer identification and allows for differentiation of benign versus malignant ovarian masses and early- and late-stage cancers.

Tumor-derived exosomes as mediators of disease and potential diagnostic biomarkers.

Tumor cells release membranous structures, defined as microvesicles or exosomes, consisting of an array of macromolecules derived from the originating cells, including proteins, lipids, and RNA. The expression of antigenic molecules recognizable by T cells originally suggested a role for these vesicles as a cell-free antigen source for anti-cancer vaccines; however, evidence demonstrates that tumor exosomes can exert a broad array of detrimental effects on the immune system - ranging from apoptosis of activated cytotoxic T cells to impairment of monocyte differentiation into dendritic cells, to induction of myeloid-suppressive cells. Immunosuppressive exosomes of tumor origin can be found in neoplastic lesions and biologic fluids from cancer patients, implying a potential role of these pathways in in vivo tumor progression and systemic paraneoplastic syndromes. Through the expression of molecules involved in angiogenesis promotion, stromal remodeling, signaling pathway activation through growth factor/receptor transfer, chemoresistance, and intercellular genetic exchange, tumor exosomes could represent a central mediator of the tumor microenvironment. Their release by tumor cells may represent the future for targeting therapeutic interventions and for development of multiplexed diagnostic biomarkers.

Patient-derived tumor-reactive antibodies as diagnostic markers for ovarian cancer.

OBJECTIVE: Most ovarian cancers are diagnosed at advanced stage (67%) and prospects for significant improvement in survival reside in early diagnosis. Our objective was to validate our array assay for the identification of ovarian cancer based on quantitation of tumor-reactive IgG. METHODS: The diagnostic array utilizes specific exosome-derived antigens to detect reactive IgG in patients' sera. Specific protein targets were isolated by immunoaffinity from exosomes derived from ovarian tumor cell lines. Sera were obtained from age-matched female volunteers, women with benign ovarian disease and with ovarian cancer. Immunoreactivity was also compared between exosomal proteins and their recombinant counterparts. RESULTS: Sera from ovarian cancer patients exhibited significantly greater immunoreactivities than either normal controls or women with benign disease (both considered negative to all antigens tested). Reactivities with nucleophosmin, cathepsin D, p53, and SSX common antigen for patients with all stages of ovarian cancer were significantly higher than for controls and women with benign ovarian disease. Reactivity with placental type alkaline phosphatase, TAG 72, survivin, NY-ESO-1, GRP78, and Muc16 (CA125) allowed the differentiation between Stage III/IV and early stage ovarian cancer. CONCLUSIONS: The quantitation of circulating tumor-reactive IgG can be used to identify the presence of ovarian cancer. The analyses of IgG recognition of specific exosomal antigens allows for the differentiation of women with benign ovarian masses from ovarian cancer, as well as distinguishing early and late stage ovarian cancers. Thus, the quantitative assessment of IgG reactive with specific tumor-derived exosomal proteins can be used as diagnostic markers for ovarian cancer.

Gene expression profiling in response to estradiol and genistein in ovarian cancer cells.

BACKGROUND: Despite optimal primary treatment of ovarian cancer, overall prognosis is poor due to recurrences. While steroid hormone receptors are frequently expressed, the role of estrogen receptor (ER) in ovarian carcinogenesis, response to treatment or prognosis has not been established. We analyzed the gene-expression in response to estradiol (E2) and genistein (Gen) in ovarian cancer cells. MATERIALS AND METHODS: Cell lines (Br-1, UL-1; Oy-1), treated with E2 (10 nM) or Gen (5 microM), were used for gene expression profiling. RT-PCR and Western immunoblotting were used to further analyze gene expression data. RESULTS: Twenty-four genes were differentially regulated in ovarian cancer cell lines. C3, CLU, COL6A1, DLC1, NME1, NRIP1, PTEN, RAC2, S100A2 were down-regulated with E2 in Br-1 and UL-1 cells. MK167, SERPINB5, SLC7A5, CDK1NA, LCN2, PLAU, PHB2, CTSB, EGLN2, ERBB2, HMGB1, ID2, ITGB4, TOP2A were up-regulated in Oy-1 cells with E2 and/or genistein. ERBB2 and ID2 (E2 and Gen), LCN2, PHB2 and HMGB1 (Gen) were down-regulated in Br-1 cells. ERalpha and ERbeta were detected in all cell lines at different levels. CONCLUSION: Variable response of ovarian cancer cells to E2 and Gen was observed. Study of ERs including splice variants, co-regulatory molecules are necessary to understand the relevance of receptors.

Alterations of T cell activation signalling and cytokine production by postmenopausal estrogen levels.

BACKGROUND: Immunosenescence is an age-associated disorder occurring primarily in T cell compartments, including altered subset composition, functions, and activation. In women, evidence implicates diminished estrogen in the postmenopausal period as a contributing factor to diminished T cell responsiveness. Since hypoestrogenism is present in postmenopausal women, our objective focused on whether T cell activation, defined as signalling molecule expressions and activation, and function, identified as IL-2 production, were affected by low estrogen. METHODS: Using Jurkat 6.1 T cells, consequences of 4 pg/ml (corresponding to postmenopausal levels) or 40 pg/ml (premenopausal levels) of estradiol (E(2)) were analyzed on signalling proteins, CD3-zeta, JAK2, and JAK3, determined by Western immunoblotting. These consequences were correlated with corresponding gene expressions, quantified by real time-polymerase chain reaction. Tyrosine phosphorylation of CD3-zeta was defined by immunoprecipitation and western immunoblotting following activation by T cell receptor (TcR) cross-linking. CD3-zeta expression and modulation was also confirmed in T cells from pre- and postmenopausal women. To assess functional consequences, IL-2 production, induced by PMA and ionomycin, was determined using enzyme-linked immunosorbent spot assay (ELISpot). RESULTS: At 40 pg/ml E(2), the level of signalling protein CD3-zeta was elevated 1.57-fold, compared with cells exposed to 4 pg/ml E(2). The CD3-zeta proteins also exhibited altered levels of activation-induced phosphorylation in the presence of 40 pg/ml E(2) versus 4 pg/ml: 23 kD phosphorylated form increased 2.64-fold and the 21 kD form was elevated 2.95-fold. Examination of kinases associated with activation signalling also demonstrated that, in the presence of 40 pg/ml E(2), JAK2 protein expression was increased 1.64-fold (p < 0.001) and JAK3 enhanced 1.79-fold (p < 0.001) compared to 4 pg/ml. mRNA levels for CD3-zeta, JAK2, and JAK3 were significantly increased following exposure to 40 pg/ml E(2) (2.39, 2.01, and 2.21 fold, respectively) versus 4 pg/ml. These findings were confirmed in vivo, since T cells from postmenopausal women exhibited 7.2-fold diminished CD3-zeta expression, compared to pre-menopausal controls and this expression was elevated 3.8-fold by addition of 40 pg/ml E(2). Functionally, Jurkat cells exposed to 40 pg/ml E(2) and activated exhibited significantly elevated numbers of IL-2 producing colonies compared to 4 pg/ml (75.3 +/- 2.2 versus 55.7 +/- 2.1 colonies, p < 0.0001). CONCLUSION: Jurkat T cells exposed to 4 pg/ml E(2) expressed significantly diminished activation signalling proteins, correlating with reduced IL-2 production. Lower signalling protein levels appear to result from decreased CD3-zeta, JAK2, and JAK3 gene expressions. These findings may provide a molecular basis for immunosenescence associated with the postmenopausal state.

Exosomal microRNA: a diagnostic marker for lung cancer.

PURPOSE: To date, there is no screening test for lung cancer shown to affect overall mortality. MicroRNAs (miRNAs) are a class of small noncoding RNA genes found to be abnormally expressed in several types of cancer, suggesting a role in the pathogenesis of human cancer. PATIENTS AND METHODS: We evaluated the circulating levels of tumor exosomes, exosomal small RNA, and specific exosomal miRNAs in patients with and without lung adenocarcinoma, correlating the levels with the American Joint Committee on Cancer (AJCC) disease stage to validate it as an acceptable marker for diagnosis and prognosis in patients with adenocarcinoma of the lung. RESULTS: To date, 27 patients with lung adenocarcinoma AJCC stages I-IV and 9 controls, all aged 21-80 years, were enrolled in the study. Small RNA was detected in the circulating exosomes. The mean exosome concentration was 2.85 mg/mL (95% CI, 1.94-3.76) for the lung adenocarcinoma group versus 0.77 mg/mL (95% CI, 0.68-0.86) for the control group (P < .001). The mean miRNA concentration was 158.6 ng/mL (95% CI, 145.7-171.5) for the lung adenocarcinoma group versus 68.1 ng/mL (95% CI, 57.2-78.9) for the control group (P < .001). Comparisons between peripheral circulation miRNA-derived exosomes and miRNA-derived tumors indicated that the miRNA signatures were not significantly different. CONCLUSION: The significant difference in total exosome and miRNA levels between lung cancer patients and controls, and the similarity between the circulating exosomal miRNA and the tumor-derived miRNA patterns, suggest that circulating exosomal miRNA might be useful as a screening test for lung adenocarcinoma. No correlation between the exosomal miRNA levels and the stage of disease can be made at this point.