Concizumab, an anti-tissue factor pathway inhibitor antibody, induces increased thrombin generation in plasma from haemophilia patients and healthy subjects measured by the thrombin generation assay.

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 concentration-dependent 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 µg kg-1 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® 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.

Streptococcus pneumoniae: proteomics of surface proteins for vaccine development.

Two formulations of pneumococcal vaccines are currently available to prevent invasive disease in adults and children. However, these vaccines will not protect against the majority of Streptococcus pneumoniae serotypes. The use of highly conserved cell-wall-associated proteins in vaccines may circumvent this problem. A proteomics approach was used to identify 270 S. pneumoniae cell-wall-associated proteins, which were then screened in a process that included in-silico, in-vitro and in-vivo validation criteria. Five potential candidates for inclusion in a vaccine were selected, expressed in Escherichia coli, and purified for use in immunisation experiments. These proteins were detected in at least 40 different serotypes of S. pneumoniae, and were expressed in S. pneumoniae isolates causing infection. Two of the five candidate proteins, the putative lipoate protein ligase (Lpl) and the ClpP protease, resulted in a reduced CFU titre and a trend towards reduced mortality in an animal sepsis model for investigating new S. pneumoniae protein vaccines.

The parasitic ciliate Ichthyophthirius multifiliis induces expression of immune relevant genes in rainbow trout, Oncorhynchus mykiss (Walbaum).

Abstract 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.

Expression of pro-inflammatory cytokines in rainbow trout (Oncorhynchus mykiss) during an infection with Ichthyophthirius multifiliis.

Inflammatory processes are known to take place in the skin of fish during an infection with the parasitic ciliate Ichthyophthirius multifiliis. The present study shows that the pro-inflammatory cytokines interleukin 1 beta (IL-1beta) and tumor necrosis factor alpha (TNF-alpha), the chemokine interleukin 8 (IL-8) and the type II IL-1 receptor (IL-1RII) are expressed in rainbow trout during an infection. In the skin an up to 17.8 times increase in the expression level of IL-1beta relative to uninfected controls was seen 4 days after infection. Upregulated expression of IL-1beta was likewise observed in the head kidney and spleen during the infection although to a lesser degree. TNF-alpha expression was seen in all examined tissues, with an increase in transcript level in the skin of up to 3.2 times the level in uninfected fish. A significant increase in the expression of IL-8 was evident in the skin and also to a lower extent in the head kidney and spleen. Expression of the down regulatory IL-1RII was seen in all three tissues. The potential involvement of the expression of these genes and their relevance in an infection with I. multifiliis are discussed.

Host responses against the fish parasitizing ciliate Ichthyophthirius multifiliis.

Recent studies have shown that fish are able to mount protective immune responses against various parasites. One of the best characterized parasite-host system in this context is the ciliate Ichthyophthirius multifiliis (Ich) parasitizing a range of freshwater fishes. Both specific and non-specific host defence mechanisms are responsible for the protection of fish against challenge infections with this ciliate. The specific humoral components comprise at least specific antibodies. The non-specific humoral elements included are the alternative complement pathway and probably lectins. Cellular factors involved in the specific response are B-cells and putative T-cells. The non-specific effector cells recognized are various leukocytes. In addition, goblet-cells and mast cells (EGC-cells) may have a function. The NCC-cell (suggested analogue to NK-cells in mammals) seems to play a role in the non-specific response. This well documented protective response in freshwater fishes against Ich has urged the development of anti-parasitic vaccines. Indeed, such products based on formalin killed parasites have been developed and found to offer the vaccinated host a satisfactory protection. However, the collection of parasites for vaccine production is extremely laborious. It involves keeping infected fish due to the fact that in vitro propagation of the parasite is still insufficiently developed. Gaining knowledge of amino acid sequences and its encoding DNA-sequences for the protective antigens (i-antigens) in the parasite was a major breakthrough. That achievement made it possible to produce a recombinant protein in E. coli and preliminary results indicated a certain protection of fish vaccinated with this product. Recent work has shown that the free-living and easily cultivated ciliate Tetrahymena can be transformed and express the i-antigen. This path seems to be promising for future development of vaccines against Ich. A novel approach in fish is the development of DNA-vaccines. Successful DNA-vaccination trials have been conducted in fish against viral infections and the technology also makes it possible to develop a DNA-vaccine against Ich. Other approaches to immuno-protection against Ich have been the use of heterologous vaccines. Thus, both bath and injection vaccination using live or killed (un-transformed) Tetrahymena has been reported to offer treated fish a certain level of protection. Such protection could be explained by non-specific reactions and the efficacy and duration of this vaccination type should be further evaluated.

Associations between epidermal thionin-positive cells and skin parasitic infections in brown trout Salmo trutta.

The dynamics of the densities of epidermal thionin-positive cells (putative mast cells) in the skin of brown trout fry were investigated during experimental infections with the skin parasites Ichthyophthirius multifiliis (Ciliophora) and Gyrodactylus derjavini (Monogenea). It was shown that the metachromatic thionin-stained cells were extremely sensitive to parasite exposure, as the density of cells in the skin of trout decreased markedly after exposure to the pathogens. As early as 7 d post infection the cell counts were significantly reduced and almost totally depleted following 9 d infection, which suggests that degranulation of the cells occurs following parasite exposure. No recruitment of new cells was seen during the study period. Some reduction in uninfected control groups indicates that the putative mast cells are sensitive to stress as well. A notable variation in densities of thionin-stained cells between different fins was found and the corneal surface was devoid of these cells. The possible implications of these cells in host-parasite interactions are suggested and discussed.

Partial cross protection against Ichthyophthirius multifiliis in Gyrodactylus derjavini immunized rainbow trout.

Partial cross protection against a skin-parasitic ciliate has been recorded in rainbow trout previously immunized with an ectoparasitic platyhelminth. The susceptibility to infection by Ichthyophthirius multifiliis differed significantly between naive and Gyrodactylus derjavini immunized rainbow trout. Fish partly immune to the ectoparasitic monogenean G. derjavini became less infected and experienced lower mortality than naive fish when exposed to I. multifiliis infections. In vitro studies on immobilization of theronts using decomplemented (heat-inactivated) serum from G. derjavini immune or non-immune hosts showed no immobilization. However, untreated serum from both immune and non-immune fish containing intact complement immobilized theronts (titre 128-256). In addition, non-specific priming of the host response with interleukin (IL-1), bacterial lipopolysaccharide (LPS), concanavalin A (Con A) or mannan did confer a partial resistance to I. multifiliis infection. This will suggest that non-specific factors including complement could be partly responsible for the host response against infections with this ciliate.