Naturally occurring CD4+ CD25+ cells in modulating immune response to administered coagulation factor VIII in factor VIII-deficient mice.

CD4+ CD25+ T regulatory (Treg) cells are critical mediators of peripheral self-tolerance and immune homeostasis. In this study, we characterized the ability of naturally occurring CD4+ CD25+ cells from the wild-type mice to modulate the immune response to administered coagulation factor VIII (FVIII) in FVIII-deficient mice. For the cell therapy, CD4+ CD25+ cells and CD4+ CD25- cells were purified from the spleens of wild-type normal mice and administered to FVIII-deficient mice prior to four injections of recombinant FVIII (rFVIII). The titre of FVIII antibodies and antibodies with inhibitory activity against FVIII was lower in the mice treated with natural CD4+ CD25+ cells or CD4+ CD25- cells compared with the mice treated only with rFVIII. We also demonstrated that CD4+ CD25- cells could differentiate to acquire the Treg phenotype expressing CD25 and FoxP3 if stimulated in vitro. These observations provide evidence that Treg cells can be used for designing cell therapy for controlling the immune response to FVIII.

The von Willebrand factor collagen-binding activity assay: clinical application.

A collagen type III based collagen-binding assay was developed for measuring the functional activity of the von Willebrand factor. The assay had a low coefficient of variance (4.8%) for normal values under optimized conditions. The results of the collagen-binding activity (CBA) assay correlated with ristocetin cofactor activity tested in normal plasma samples (n=29). We found that the CBA of blood group O is lower than that of other blood groups. The test was used for the diagnosis of von Willebrand's disease (VWD) and for estimating the response to treatment with DDAVP (1-deamino-D-arginine-8 vasopressin) and factor VIII concentrate. A mean ratio of VWF antigen (VWF:Ag) to CBA of 1.5 indicated type 1 and of 2.7 indicated type 2 VWD. The increase in the collagen-binding activity of VWF released in type 1 VWD patients (n=7) after treatment with DDAVP was higher than the increase in the VWF antigen; this is characteristic of very high multimers with greater functional activity. Factor VIII concentrate Koate-HP (Bayer) administered to a patient with VWD type 3 had a mean residence time of 12.6 h for VWF:Ag and 11.2 h for CBA. These findings suggest that the collagen-binding assay is a useful test for measuring the functional activity of VWF in plasma samples, factor VIII concentrates, as well as for estimating the outcome of treatment.

von Willebrand factor in factor VIII concentrates protects against neutralization by factor VIII antibodies of haemophilia A patients.

We investigated the neutralization activity of factor VIII (FVIII) antibodies of 12 haemophilia A patients, acquired during treatment with plasma-derived FVIII concentrates. All plasma samples, drawn in a clinically stable situation before any immunotolerance treatment, contained anti-A2 domain and anti-light-chain FVIII antibodies. In nine patients' plasmas, containing relatively high amounts of FVIII light-chain antibodies (53-96%), a higher neutralization activity was found against recombinant FVIII concentrate (Recombinate) than against plasma-derived von Willebrand factor (vWF)-containing concentrate (Haemoctin SDH). No difference in neutralization of the two concentrates was found in two patients' plasmas with almost equal content of FVIII light- and heavy-chain antibodies, or one plasma with predominantly heavy-chain antibodies. These results suggest that haemophilia A patients with relatively high amounts of FVIII light-chain antibodies in plasma might benefit by infusion of FVIII concentrates containing vWF because vWF appears to have some protective effect on FVIII. This hypothesis should be tested by a clinical study.