A serological method for detection of Nosema ceranae.

AIMS: We developed a new method for detection of the intracellular parasite, Nosema ceranae, one of the most economically devastating pathogens of the honeybee. METHODS AND RESULTS: The SWP-32 antibody was used for the development of an enzyme-linked immunosorbent assay (ELISA). We also compared the efficiency of this ELISA to microscopy and quantitative real-time (qRT) PCR, the methods currently in use. CONCLUSIONS: ELISA is comparable in sensitivity with the qRT-PCR, less expensive and faster. When this method is commercialized and made available to bee-keepers, it will allow them to make informed decisions for the application of in-hive chemicals. Hence, bee-keepers may be able to determine when treatments for control of N. ceranae are unnecessary and reduce the cost, time and possible side effects of these treatments. SIGNIFICANCE AND IMPACT OF THE STUDY: This assay provides the first serological method for detection of N. ceranae in bee colonies, which is as sensitive as DNA amplification. It can be easily adopted for both laboratory and field applications.

Adhesive properties of the isolated amino-terminal domain of platelet glycoprotein Ibalpha in a flow field.

We have examined the interaction between the amino-terminal domain of platelet glycoprotein (GP) Ibalpha and immobilized von Willebrand Factor (vWF) under flow conditions in the absence of other components of the GP Ib-IX-V complex. Latex beads were coated with a recombinant fragment containing GP Ibalpha residues 1-302, either with normal sequence or with the single G233V substitution that causes enhanced affinity for plasma vWF in platelet-type pseudo-von-Willebrand disease. Beads coated with native fragment adhered to vWF in a manner comparable to platelets, showing surface translocation that reflected the transient nature of the bonds formed. Thus, the GP Ibalpha extracellular domain is necessary and sufficient for interacting with vWF under high shear stress. Beads coated with the mutated fragment became tethered to vWF in greater number and had lower velocity of translocation than beads coated with the normal counterpart, suggesting that the G233V mutation lowers the rate of bond dissociation. Our findings define an approach for studying the biomechanical properties of the GP Ibalpha-vWF bond and suggest that this interaction is tightly regulated to allow rapid binding at sites of vascular injury, while permitting the concurrent presence of receptor and ligand in the circulation.

Contribution of distinct adhesive interactions to platelet aggregation in flowing blood.

Aggregation of blood platelets contributes to the arrest of bleeding at sites of vascular injury, but it can occlude atherosclerotic arteries and precipitate diseases such as myocardial infarction. The bonds that link platelets under flow conditions were identified using confocal videomicroscopy in real time. Glycoprotein (GP) Ibalpha and von Willebrand factor (vWF) acted in synergy with alphaIIbbeta3 and fibrinogen to sustain platelet accrual at the apex of thrombi where three-dimensional growth resulted in increasing shear rates. The specific function of distinct adhesion pathways in response to changing hemodynamic conditions helps to explain hemostatic and thrombotic processes.

Distinct mechanisms of platelet aggregation as a consequence of different shearing flow conditions.

Platelet aggregation contributes to arresting bleeding at wound sites, but may cause occlusion of atherosclerotic vessels, thus curtailing blood flow to vital organs. According to current dogma, the integrin alphaIIbbeta3 plays an exclusive role in linking platelets to one another through interactions with fibrinogen or vWf. We demonstrate here that, depending on shearing flow conditions, this process may require vWf binding to glycoprotein Ibalpha, even when alphaIIbbeta3 is competent to bind adhesive ligands. Platelet activation induced solely by high shear stress is initiated by glycoprotein Ibalpha interaction with vWf, but results in aggregation only if the latter can bind concurrently to alphaIIbbeta3. In contrast, platelets exposed to high shear rate after activation by exogenous agonists such as ADP and epinephrine can aggregate when fibrinogen is the alphaIIbbeta3 adhesive ligand, yet only if vWf binding to glycoprotein Ibalpha can also occur. Thus, the latter interaction appears to provide a bond with biomechanical properties necessary to overcome the effects of high shear rate and initiate interplatelet cohesion. These findings highlight the distinct function of two adhesive receptors mediating platelet aggregation under varying fluid dynamic conditions, and modify the current interpretation of a crucial event in hemostasis and thrombosis.

Role of beta3 integrins in melanoma cell adhesion to activated platelets under flow.

Mechanisms mediating tumor cell attachment to the vessel wall under flow conditions are largely unknown. Therefore we analyzed the ability of human melanoma cells to adhere to an immobilized matrix during blood flow and determined the role of platelets in this process. In a parallel plate flow chamber, M21 melanoma cells were suspended in human blood and perfused over a collagen I matrix at a wall shear rate of 50 s-1 (2 dynes/cm2) to simulate venous flow over a thrombogenic surface. Melanoma cell interaction with the matrix or blood cells and platelets was monitored and quantified by fluorescence and confocal laser microscopy. Despite their ability to adhere to collagen I under static conditions, M21 cells failed to attach directly to this matrix during blood flow. However, they associated with adherent thrombi, and this resulted in stable melanoma cell arrest. Inhibition of platelet activation or platelet integrin alphaIIbbeta3 function abolished M21 cell attachment. Melanoma cell interaction with thrombi was specific and required beta3 integrin expression. M21-L cells which lack integrin alphavbeta3 failed to associate with thrombi and to arrest during blood flow. Transfection of these cells with the integrin subunits alphav or alphaIIb resulted in variants expressing alphavbeta3, as in the wild type, or alphaIIbbeta3. Both variants were able to associate with thrombi and to arrest during blood flow. Therefore, beta3 integrin-mediated binding to activated platelets represents an efficient mechanism for melanoma cell arrest under flow, and this may contribute to the role of platelets in hematogenous metastasis.

Initiation of platelet adhesion by arrest onto fibrinogen or translocation on von Willebrand factor.

We have identified two distinct mechanisms initiating the adhesion of flowing platelets to thrombogenic surfaces. The intergrin alpha IIb beta 3 promotes immediate arrest onto fibrinogen but is fully efficient only at wall shear rates below 600-900 s-1, perhaps because of a relatively slow rate of bond formation or low resistance to tensile stress. In contrast, glycoprotein Ib alpha binding to immobilized von Willebrand factor (vWF) appears to have fast association and dissociation rates as well as high resistance to tensile stress, supporting slow movement of platelets in continuous contact with the surface even at shear rates in excess of 6000 s-1. This eventually allows activated alpha IIb beta 3 to arrest platelets onto vWF under conditions not permissive of direct binding to fibrinogen. The coupling of these different functions may be crucial for thrombogenesis.

Interleukin-1 beta induces differential adhesiveness on human endothelial cell surfaces.

The adhesive strength between HL-60 and endothelial cells activated with IL-1 beta was investigated according to the region (area) on the endothelial cell surface where the HL-60 cell was attached (nuclear, junctional, or cytoplasmic). Using a micropipette single-cell manipulation system, we demonstrated that the increase of adhesive force was due to the activation of the endothelial cell with IL-1 beta and a function of the region of the endothelial cell. The increase in adhesion strength due to the increased expression of E-Selectin on the endothelial cell was studied by the addition of monoclonal antibodies against E-Selectin, ICAM-1, VCAM, and PECAM-1. Our findings suggest that the greatest adhesive strength was seen in the junctional region where the antibodies could not block the adhesion. However, the contribution of E-Selectin was significant in the nuclear region.