Catch of the Day: New Serum Amyloid A (SAA) Antibody Is a Valuable Tool to Study Fish Health in Salmonids.

Serum amyloid A (SAA) proteins belong to a family of acute-phase reactants, playing an integral role in defending the organism from pathological damage. Despite a wealth of data on the regulation of SAA transcripts in teleosts, there is only limited information on these proteins' abundance in fish. The aim of this study is to characterise SAA protein levels in salmonids using a newly developed antibody specific to salmonid SAA. The salmonid SAA antibody detected SAA and accurately discriminated between stimulated and control specimens from rainbow trout macrophage cell line (RTS-11) in vitro, as well as rainbow trout challenged with Aeromonas salmonicida- or flagellin-stimulated Atlantic salmon in vivo. The presence of SAA protein was analysed in RTS-11 cell line supernatants, liver, and spleen samples using ELISA, immunoblotting, and immunohistochemistry. This study is the first to characterise SAA protein levels in salmonids in vivo and in vitro. The newly developed salmonid SAA antibody was able to discriminate between stimulated and unstimulated specimens, showing that it can be used to study the acute-phase response in salmonids with the potential to be further developed into assays to monitor and evaluate health in wild and farmed fish.

ABCG2 Is Overexpressed on Red Blood Cells in Ph-Negative Myeloproliferative Neoplasms and Potentiates Ruxolitinib-Induced Apoptosis.

Myeloproliferative neoplasms (MPNs) are a group of disorders characterized by clonal expansion of abnormal hematopoietic stem cells leading to hyperproliferation of one or more myeloid lineages. The main complications in MPNs are high risk of thrombosis and progression to myelofibrosis and leukemia. MPN patients with high risk scores are treated by hydroxyurea (HU), interferon-α, or ruxolitinib, a tyrosine kinase inhibitor. Polycythemia vera (PV) is an MPN characterized by overproduction of red blood cells (RBCs). ABCG2 is a member of the ATP-binding cassette superfamily transporters known to play a crucial role in multidrug resistance development. Proteome analysis showed higher ABCG2 levels in PV RBCs compared to RBCs from healthy controls and an additional increase of these levels in PV patients treated with HU, suggesting that ABCG2 might play a role in multidrug resistance in MPNs. In this work, we explored the role of ABCG2 in the transport of ruxolitinib and HU using human cell lines, RBCs, and in vitro differentiated erythroid progenitors. Using stopped-flow analysis, we showed that HU is not a substrate for ABCG2. Using transfected K562 cells expressing three different levels of recombinant ABCG2, MPN RBCs, and cultured erythroblasts, we showed that ABCG2 potentiates ruxolitinib-induced cytotoxicity that was blocked by the ABCG2-specific inhibitor KO143 suggesting ruxolitinib intracellular import by ABCG2. In silico modeling analysis identified possible ruxolitinib-binding site locations within the cavities of ABCG2. Our study opens new perspectives in ruxolitinib efficacy research targeting cell types depending on ABCG2 expression and polymorphisms among patients.

Altered Ca2+ Homeostasis in Red Blood Cells of Polycythemia Vera Patients Following Disturbed Organelle Sorting during Terminal Erythropoiesis.

Over 95% of Polycythemia Vera (PV) patients carry the V617F mutation in the tyrosine kinase Janus kinase 2 (JAK2), resulting in uncontrolled erythroid proliferation and a high risk of thrombosis. Using mass spectrometry, we analyzed the RBC membrane proteome and showed elevated levels of multiple Ca2+ binding proteins as well as endoplasmic-reticulum-residing proteins in PV RBC membranes compared with RBC membranes from healthy individuals. In this study, we investigated the impact of JAK2V617F on (1) calcium homeostasis and RBC ion channel activity and (2) protein expression and sorting during terminal erythroid differentiation. Our data from automated patch-clamp show modified calcium homeostasis in PV RBCs and cell lines expressing JAK2V617F, with a functional impact on the activity of the Gárdos channel that could contribute to cellular dehydration. We show that JAK2V617F could play a role in organelle retention during the enucleation step of erythroid differentiation, resulting in modified whole cell proteome in reticulocytes and RBCs in PV patients. Given the central role that calcium plays in the regulation of signaling pathways, our study opens new perspectives to exploring the relationship between JAK2V617F, calcium homeostasis, and cellular abnormalities in myeloproliferative neoplasms, including cellular interactions in the bloodstream in relation to thrombotic events.