Splenectomy in zebrafish: a new model for immune thrombocytopenia

In humans, splenectomy is performed to treat many clinical disorders, including immune thrombocytopenia. However, the incidence of splenectomies for immune thrombocytopenia as a therapeutic has significantly declined over the past decade due to the availability of new therapies. Infection and sepsis as a result of splenectomies are well documented, but other long-term effects are not well characterized. Evidence suggests that persons who have had a prior splenectomy may be at an increased risk of vascular conditions. Also, elevated levels of cell-derived microparticles appear to contribute to an increased risk of thrombosis and cardiovascular disease. However, in vivo studies on the increased levels of microparticles following splenectomy are limited. In order to understand the effects of splenectomies, we developed a protocol for splenectomy in adult zebrafish. After anesthesia, the spleen was removed under a stereomicroscope after making an incision on the ventral side of the fish. The spleen was removed by pulling with forceps. The incision was closed by Vetbond tissue glue. Blood collected from both splenectomized zebrafish and those that underwent sham surgeries was immunolabeled with polyclonal antisera against αIIb, followed by flow cytometry. We observed elevated levels of thrombocytes and their microparticles in splenectomized zebrafish. Finally, by injecting αIIb antibody intravenously into zebrafish, we found the thrombocyte counts decreased, suggesting the fish developed immune thrombocytopenia like conditions, which were then reversed by splenectomy. In summary, the model developed here should be useful to study molecular changes due to splenectomy. Also, the zebrafish will be useful in modeling treatment of immune thrombocytopenia like conditions

Trout thrombocytes aggregate to collagen in a Src dependent manner.

<p>Anticoagulated whole blood from rainbow trout was diluted 1∶1 with normal saline (0.9%) and stirred for 3 min at room temperature in a Multiplate mini-cuvette. Dasatinib (5 µM) or DMSO vehicle control was pre-incubated at room temperature for 1 min followed by addition of collagen, 30 or 10 µg/ml, and aggregation was monitored for 12 min at room temperature. Aggregation was measured in arbitrary impedance units (AU).</p

G6fL responds to collagen in a cell line assay through its ITAM.

<p>DT40 cells were transfected with wild type zebrafish G6fL (A) or with point mutants of G6fL (Y499F, Y515F and Y527F) (B) and stimulated with either collagen (10 µg/ml) or CRP (3 µg/ml). Where stated, cells were pre-incubated with 20 µM PP2. 6 hrs following stimulation, cells were lysed and luciferase activity was measured as a readout of signalling. Data is expressed as fold over basal (dotted line). Statistical significance was calculated with a Student’s t-test (*P>0.05, ***P>0.005). Similar levels of expression of all G6fL constructs was confirmed by western blotting (not shown).</p

Trout thrombocytes adhere to and spread on collagen in a Src dependent manner.

<p>A) Trout blood was diluted by a factor of 100 to reduce the number of cells, and then allowed to adhere to either collagen or fibrinogen coated coverslips for 90 min at room temperature, either in the presence of 20 µM dasatinib or DMSO vehicle control. B) Anticoagulated whole blood was flowed through collagen coated glass capillary tubes at a range of shear rates (i) or at 100 s⁻¹ for inhibitor studies (ii). Blood was pre-treated with either 20 µM dasatinib or DMSO vehicle control.</p

Foreign aid, poverty reduction, and democracy

Eradication of poverty is the most pervasive goal of donors' foreign aid programmes. As a result, there has been much research on the degree of correlation between aid and poverty reduction. However, this work to date has shed little light on the direction of causation between the two variables. Using the method of Granger causality, and conditioning aid and poverty on the state of democracy in developing countries, this study asks whether aid flows impact poverty, whether poverty influences aid flows, or whether causality proceeds in both directions simultaneously. While the results identify no causal relationships in some of the sub-samples, they point to the existence of a multitude of relationships across others.

G6f-like is an ITAM-containing collagen receptor in thrombocytes

Collagen activates mammalian platelets through a complex of the immunoglobulin (Ig) receptor GPVI and the Fc receptor γ-chain, which has an immunoreceptor tyrosine-based activation motif (ITAM). Cross-linking of GPVI mediates activation through the sequential activation of Src and Syk family kinases and activation of PLCγ2. Nucleated thrombocytes in fish are activated by collagen but lack an ortholog of GPVI. In this study we show that collagen activates trout thrombocytes in whole blood and under flow conditions through a Src kinase driven pathway. We identify the Ig receptor G6f-like as a collagen receptor and demonstrate in a cell line assay that it signals through its cytoplasmic ITAM. Using a morpholino for in vivo knock-down of G6f-like levels in zebrafish, we observed a marked delay or absence of occlusion of the venous and arterial systems in response to laser injury. Thus, G6f-like is a physiologically relevant collagen receptor in fish thrombocytes which signals through the same ITAM-based signalling pathway as mammalian GPVI, providing a novel example of convergent evolution