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During an infection with the parasitic ciliate Ichthyophthirius multifiliis, expression of genes encoding complement factor C3, inducible nitric oxide synthase (iNOS), immunoglobulin (IgM) and major histocompatibility complex (MHC-II) was examined in the skin, head kidney and spleen of rainbow trout using semi-quantitative reverse transcriptase-polymerase chain reaction. Induction of C3 transcription levels was evident in the skin and spleen showing extra-hepatic production of C3. MHC-II and IgM levels were increased in both head kidney and skin suggesting a production of antibodies at the site of infection, as well as in the lymphoid organs. iNOS expression was only increased briefly in the skin during the infection. These data suggest that complement is involved in immune reactions against I. multifiliis and that mucosal antibodies might be produced at the site of infection.

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Aims: Concizumab, a humanized monoclonal antibody against tissue factor pathway inhibitor (TFPI), is being developed as a subcutaneously (s.c.) administered treatment for haemophilia. It demonstrated a concentrationdependent procoagulant effect in functional TFPI assays; however, global haemostatic assays, such as the thrombin generation assay (TGA), offer a more complete picture of coagulation. We investigated how concizumab affects thrombin generation following ex vivo spiking in plasma from haemophilia patients using the TGA, and if the assay can detect the effect of multiple s.c. concizumab doses in healthy subjects. Methods: For the ex vivo spiking study, platelet-poor plasma (PPP) from 18 patients with severe haemophilia was spiked with 0.001-500 nM concizumab. For the multiple-dosing study, four healthy males received concizumab 250 lg kg À1 s.c. every other day for eight doses; blood was collected before and after dosing and processed into PPP. In both studies, thrombin generation was measured using a Calibrated Automated Thrombogram â system with 1 pM tissue factor. Results: In spiked samples from haemophilia patients, peak thrombin and endogenous thrombin potential (ETP) increased concentration dependently, reaching near-normal levels at concizumab concentrations >10 nM. Repeated s.c. doses of concizumab in healthy subjects increased both peak thrombin and ETP; these effects were sustained throughout the dosing interval. Conclusions: Thrombin generation assay demonstrated increased thrombin generation with concizumab after ex vivo spiking of haemophilia plasma and multiple s.c. doses in healthy subjects, supporting both the utility of the TGA in evaluating concizumab treatment and the potential of s.c. concizumab as a novel haemophilia therapy.

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