Levels Of Microvesicles Research Articles

Increased serum levels of microvesicles in nonvalvular atrial fibrillation determinated by ELISA using a specific monoclonal antibody AD-1

Microvesicles are involved in different pathological processes such as inflammation, coagulation and tumor progression. We intended to establish an immunoaffinity capture method for detecting microvesicles and bioactive effectors carried on them using a specific homemade monoclonal antibody AD-1. By this method we investigated the association of inflammation with platelet activation in patients with nonvalvular atrial fibrillation (NVAF). A case-control study of 90 Chinese subjects selected in 3 groups: control, paroxysmal AF, and persistent AF. After capturing the microvesicles of serum using a specific monoclonal antibody AD-1, the amounts of LAP, IL-1β and P-selectin loaded on these microvesicles were quantified by either enzyme activity assay (LAP) or ELISA respectively. Compared with normal controls, the patients with persistent AF showed significantly increased serum levels of microvesicles (P<0.001), microvesicle-bound IL-1 β (P=0.019) and microvesicle-bound P-selectin (P=0.001). The latter two were significantly correlated with each other (r(2)=0.371, r=0.616, P<0.001). The microvesicle-bound IL-1β (β=0.570, P<0.001) and body weight (β=0.427, P=0.002) were as independent predictors of platelet activation. The method was easy and reproducible. Inflammation may be involved in the activation of platelets in NVAF.

Membrane microvesicles: Macromessengers in cancer disease and progression

Thrombotic complications have been documented in patients with cancer, and associated with tumor progression. Cancer patients have an increased level of circulating submicrometric (0.1–1 μm) membrane fragments termed microvesicles (MV) or microparticles. Variations in MV levels and phenotypes make them relevant pathogenic markers of thrombotic disorders and vascular damage. MV are released from the plasma membrane of activated or apoptotic cells, and are considered efficient effectors of the hemostatic or thrombotic responses. They are mostly characterized by the presence of procoagulant phospholipids at their surface and eventually that of tissue factor depending on the cells they originate from. These procoagulant entities allow them to initiate and propagate thrombotic reactions within the blood vessels. MV are also recognized as proximal or remote mediators of cell-to-cell communication. The mechanisms through which MV interact with target cells remain unclear although a number of studies suggest involvement of MV-cell fusion and/or ligand-receptor interactions. It has however to be emphasized that MV do not necessarily elicit deleterious responses. This review focuses on the role of MV in cancer-associated thrombosis.

Characterization of Microvesicles in B-Cell Chronic Lymphocytic Leukemia (CLL): A Potential Mediator in CLL B Cell Disease Progression?.

While cellular communication and interactive signaling typically relies on multiple mechanisms including secretion of various growth factors, cytokines, bioactive lipids, and adhesion molecules, we have recently focused on cell-to-cell communication that involves circular membrane vesicles designated as microvesicles (MVs). These are normal constituents of blood plasma released by leukocytes, endothelium, platelets and erythrocytes and are generated by cell activation and growth. In normal serum, the order of MV abundance is platelet-derived (80%), endothelial (10%) and leukocyte-derived (10%) but MV can also be released by malignant cells. The size varies from 0.1–1.0 μm and, MVs express antigens characteristic of the cell of origin, carry other membrane and cytoplasmic constituents and exhibit negatively charged phospholipids (phosphatidylserine) detectable by Annexin V binding. To begin to characterize MV and their potential for communication in CLL, we isolated and characterized MVs from the plasma of chronic lymphocytic leukemia B-cell (CLL B) patients (Rai 0-IV). After isolation from platelet-free plasma, MVs were characterized by flow cytometry for forward and side scatter using 1μm fluorescent beads and stained with Annexin V. Our results suggest that MVs are heterogeneous in size with the majority of MVs within 1μm. Transmission electron microscopy confirmed the size heterogeneity of MVs and exhibited the morphology of the membrane-bound vesicles after phosphotungstic acid staining onto parlodium-coated 300 mesh copper grids. Flow cytometric analysis further demonstrated the presence of CD61 (platelet/megakaryocyte marker), CD19, CD5, CD52, and CD20 on the surface of MV, albeit in differential amounts depending on the stage of the disease and the total leukocyte count. For example, in Rai stage IV (n= 5), we found lesser numbers of MV carrying platelet-derived marker CD61 and more CD19 (41–79%), and CD5 compared to normal MV; 1–5% MV from healthy controls expressed CD19 while Rai stage 4 patients MV expressed CD19 from 41–79%. These results suggest that a significant number of MVs are generated from the leukemic B-lymphocytes in more advanced Rai stage. However, we also found that patients with a higher blood lymphocyte count but lower Rai-stage (Rai I/II) contain moderate levels of CD19-bearing MVs (13–43%). Importantly, presence of MVs carrying CD52/CD20 in the blood of CLL patients may undermine the therapeutic effectiveness of alemtuzumab and rituximab. To this end, we observed that MVs could be incorporated into the CLL B cells as evident from the expression of the platelet antigen CD61 on CLL B cell surfaces after co-culture of CD61-bearing MVs with CLL B cells. Global proteomic analysis of MVs isolated from an advanced stage (Rai IV) CLL B patient detected the presence of about 700 proteins that normally reside in the nucleus, cytoplasm or membrane. In summary, we have isolated and characterized plasma MV from CLL B patients and found them to be more likely generated from CLL B cells in relation to disease stage or total lymphocyte count. These MVs contain a multitude of proteins and are able to integrate into bystander CLL B cells. We also found that these MVs carry a large set of proteins, which could potentially not only modulate cell-cell communication but also interrupt monoclonal antibody directed therapy. Further studies are underway to dissect their roles in CLL B malignancy.

Enhanced activation of platelets with abnormal release of RANTES in human immunodeficiency virus type 1 infection

Besides their role in hemostasis, platelets are involved in inflammatory and immunological processes, and we hypothezise that platelet activation may play an immunopathogenetic role in HIV-1 infection. Blood was drawn from 15 controls and 20 HIv-1-infected patients with normal platelet counts, classified into groups of non-AIDS and AIDS. Platelet activation was detected using flow cytometry with mAbs against the release markers P-selectin and CD63, mAb against GPIb, and the probe annexin V detecting surface exposure of aminophospholipids. The amount of microvesicles was measured using mAb against GβIIIa. Compared to controls, blood samples from HIV-1-infected patients showed significantly enhanced levels of microvesicles and activated platelets as detected by their exposure of P-selectin, CD63, and aminophospholipids, as well as reduction in GPIb expression. Increased expression of P-selectin and amounts of microvesicles were most pronounced in advanced clinical and immunological disease. When studying the effect of HIV-1 protease inhibitor therapy (indinavir) on platelet activation, we found that concomitant with a profound decrease in plasma viral load, there was a near normalization of several of the parameters reflecting enhanced platelet activation. Finally, we demonstrated that platelets may be an important source of the chemokine RANTES in HIV-1-infected patients. Although both unstimulated and SFLLRN-stimulated platelets from asymptomatic patients had enhanced release of RANTES, platelets from AIDS patients were characterized by markedly enhanced spontaneous, but decreased SFLLRN-stimulated release of this chemokine. Taken together, these results, which demonstrate for the first time increased platelet activation in HIV-1-infected patients with normal platelet counts, may represent a previously unrecognized immunopathogenic factor in HIV-1 infection.—Holme, P. A., Müller, F., Solum, N. O., Brosstad, F., Frøland, S. S., Aukrust, P Enhanced activation of platelets with abnormal release of RANTES in human immunodeficiency virus type 1 infection. FASEB J. 12, 79–90 (1998)

Enhanced activation of platelets with abnormal release of RANTES in human immunodeficiency virus type 1 infection.

Besides their role in hemostasis, platelets are involved in inflammatory and immunological processes, and we hypothesize that platelet activation may play an immunopathogenetic role in HIV-1 infection. Blood was drawn from 15 controls and 20 HIV-1-infected patients with normal platelet counts, classified into groups of non-AIDS and AIDS. Platelet activation was detected using flow cytometry with mAbs against the release markers P-selectin and CD63, mAb against GPIb, and the probe annexin V detecting surface exposure of aminophospholipids. The amount of microvesicles was measured using mAb against GPIIIa. Compared to controls, blood samples from HIV-1-infected patients showed significantly enhanced levels of microvesicles and activated platelets as detected by their exposure of P-selectin, CD63, and aminophospholipids, as well as reduction in GPIb expression. Increased expression of P-selectin and amounts of microvesicles were most pronounced in advanced clinical and immunological disease. When studying the effect of HIV-1 protease inhibitor therapy (indinavir) on platelet activation, we found that concomitant with a profound decrease in plasma viral load, there was a near normalization of several of the parameters reflecting enhanced platelet activation. Finally, we demonstrated that platelets may be an important source of the chemokine RANTES in HIV-1-infected patients. Although both unstimulated and SFLLRN-stimulated platelets from asymptomatic patients had enhanced release of RANTES, platelets from AIDS patients were characterized by markedly enhanced spontaneous, but decreased SFLLRN-stimulated release of this chemokine. Taken together, these results, which demonstrate for the first time increased platelet activation in HIV-1-infected patients with normal platelet counts, may represent a previously unrecognized immunopathogenic factor in HIV-1 infection.

Identification and ultrastructural localization of a calretinin-like calcium-binding protein (protein 10) in the guinea pig and rat inner ear

Calretinin has been identified as a brain specific calcium-binding protein which appears as a prominent protein in the cochlear nucleus. We identified and localized calretinin in the guinea pig and rat inner ear using polyclonal antibodies. Immunoblot analyses of guinea pig and rat auditory nerve homogenates revealed an immunoreactive band migrating with the same molecular weight as the purified protein, at M r = 29k . Immunocytochemistry was carried out at the light and electron microscope levels. In the guinea pig cochlea, inner hair cells, Deiters' cells, Hensen's cells and interdental cells of the spiral limbus were stained. Most of the cochlear ganglion cells were immunostained. In the guinea pig vestibular organs, the staining was exclusively neuronal and localized in large nerve fibers and nerve calices of the apex of the cristae. Only some vestibular ganglion cells were stained. In the rat cochlea, inner hair cells and most of the ganglion neurons were immunoreactive. In the rat vestibule, large nerve fibers and calices were stained as were some type II hairs cells. Only some vestibular ganglion cells were reactive. Electron microscopic observations of immunostained guinea pig cochlea and vestibule showed that the staining was cytosolic. In addition, specific sub-localization was also found in the apical portion of the nerve calices in association with microvesicles. These results describe the discrete localization of calretinin in the cochlea and in the vestibular receptors and suggest a function associated with biochemical regulations at the level of microvesicles in vestibular afferent neurons.