Ade Kallas

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.

Nocodazole Treatment Decreases Expression of Pluripotency Markers Nanog and Oct4 in Human Embryonic Stem Cells

Nocodazole is a known destabiliser of microtubule dynamics and arrests cell-cycle at the G2/M phase. In the context of the human embryonic stem cell (hESC) it is important to understand how this arrest influences the pluripotency of cells. Here we report for the first time the changes in the expression of transcription markers Nanog and Oct4 as well as SSEA-3 and SSEA-4 in human embryonic cells after their treatment with nocodazole. Multivariate permeabilised-cell flow cytometry was applied for characterising the expression of Nanog and Oct4 during different cell cycle phases. Among untreated hESC we detected Nanog-expressing cells, which also expressed Oct4, SSEA-3 and SSEA-4. We also found another population expressing SSEA-4, but without Nanog, Oct4 and SSEA-3 expression. Nocodazole treatment resulted in a decrease of cell population positive for all four markers Nanog, Oct4, SSEA-3, SSEA-4. Nocodazole-mediated cell-cycle arrest was accompanied by higher rate of apoptosis and upregulation of p53. Twenty-four hours after the release from nocodazole block, the cell cycle of hESC normalised, but no increase in the expression of transcription markers Nanog and Oct4 was detected. In addition, the presence of ROCK-2 inhibitor Y-27632 in the medium had no effect on increasing the expression of pluripotency markers Nanog and Oct4 or decreasing apoptosis or the level of p53. The expression of SSEA-3 and SSEA-4 increased in Nanog-positive cells after wash-out of nocodazole in the presence and in the absence of Y-27632. Our data show that in hESC nocodazole reversible blocks cell cycle, which is accompanied by irreversible loss of expression of pluripotency markers Nanog and Oct4.

SOX2 Is Regulated Differently from NANOG and OCT4 in Human Embryonic Stem Cells during Early Differentiation Initiated with Sodium Butyrate

Transcription factors NANOG, OCT4, and SOX2 regulate self-renewal and pluripotency in human embryonic stem (hES) cells; however, their expression profiles during early differentiation of hES cells are unclear. In this study, we used multiparameter flow cytometric assay to detect all three transcription factors (NANOG, OCT4, and SOX2) simultaneously at single cell level and monitored the changes in their expression during early differentiation towards endodermal lineage (induced by sodium butyrate). We observed at least four distinct populations of hES cells, characterized by specific expression patterns of NANOG, OCT4, and SOX2 and differentiation markers. Our results show that a single cell can express both differentiation and pluripotency markers at the same time, indicating a gradual mode of developmental transition in these cells. Notably, distinct regulation of SOX2 during early differentiation events was detected, highlighting the potential importance of this transcription factor for self-renewal of hES cells during differentiation.

Epitope specificity of anti-FVIII antibodies during immune tolerance therapy with factor VIII preparation containing von Willebrand factor

The study aimed at characterizing the putative changes in the epitope specificity of anti-FVIII antibodies during a successful immune tolerance treatment of the haemophilia A patient with the factor VIII (FVIII) preparation containing the von Willebrand factor (VWF). At the beginning of treatment, anti-FVIII inhibitory antibodies recognizing predominantly the light chain of FVIII were prevalent and persisted throughout the treatment. More detailed characterization of the FVIII antibody epitope specificity by using GST-fusion proteins corresponding to different FVIII domains revealed the prevalence of C1-domain-specific antibodies, while a remarkably lower amount of antibodies were targeted at the C2 and the a3 domains of the FVIII light chain and towards the A2 and the A1 domain of the FVIII heavy chain. The epitope specificity of antibodies remained rather unchanged throughout treatment except the elevated level of C2-domain-specific FVIII antibodies after a temporary interruption of treatment. The patients antibodies were unable to interfere with the FVIII binding to VWF or to phospholipids, but inhibited FXa generation and the binding of FX to FVIII on the phospholipid monolayer. Thus, a unique pattern of the epitope specificity of FVIII antibodies and the mechanism to inhibit FVIII:C activity by FVIII-light-chain-specific antibodies were characterized.

Assessment of the Potential of CDK2 Inhibitor NU6140 to Influence the Expression of Pluripotency Markers NANOG, OCT4, and SOX2 in 2102Ep and H9 Cells

As cyclin-dependent kinases (CDKs) regulate cell cycle progression and RNA transcription, CDKs are attractive targets for creating cancer cell treatments. In this study we investigated the effects of the small molecular agent NU6140 (inhibits CDK2 and cyclin A interaction) on human embryonic stem (hES) cells and embryonal carcinoma-derived (hEC) cells via the expression of transcription factors responsible for pluripotency. A multiparameter flow cytometric method was used to follow changes in the expression of NANOG, OCT4, and SOX2 together in single cells. Both hES and hEC cells responded to NU6140 treatment by induced apoptosis and a decreased expression of NANOG, OCT4, and SOX2 in surviving cells. A higher sensitivity to NU6140 application in hES than hEC cells was detected. NU6140 treatment arrested hES and hEC cells in the G2 phase and inhibited entry into the M phase as evidenced by no significant increase in histone 3 phosphorylation. When embryoid bodies (EBs) formed from NU6104 treated hES cells were compared to EBs from untreated hES cells differences in ectodermal, endodermal, and mesodermal lineages were found. The results of this study highlight the importance of CDK2 activity in maintaining pluripotency of hES and hEC cells and in differentiation of hES cells.

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

Summary.  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.

Changes in Laminin Expression Pattern during Early Differentiation of Human Embryonic Stem Cells.

Laminin isoforms laminin-511 and -521 are expressed by human embryonic stem cells (hESC) and can be used as a growth matrix to culture these cells under pluripotent conditions. However, the expression of these laminins during the induction of hESC differentiation has not been studied in detail. Furthermore, the data regarding the expression pattern of laminin chains in differentiating hESC is scarce. In the current study we aimed to fill this gap and investigated the potential changes in laminin expression during early hESC differentiation induced by retinoic acid (RA). We found that laminin-511 but not -521 accumulates in the committed cells during early steps of hESC differentiation. We also performed a comprehensive analysis of the laminin chain repertoire and found that pluripotent hESC express a more diverse range of laminin chains than shown previously. In particular, we provide the evidence that in addition to α1, α5, β1, β2 and γ1 chains, hESC express α2, α3, β3, γ2 and γ3 chain proteins and mRNA. Additionally, we found that a variant of laminin α3 chain—145 kDa—accumulated in RA-treated hESC showing that these cells produce prevalently specifically modified version of α3 chain in early phase of differentiation.

Von Willebrand Factor-Specific Antibodies Developing upon Treatment of FVIII-Deficient Mice with Different FVIII Preparations

tion of FVIII-specific antibodies as a response to treatment with the FVIII-VWF complex compared with FVIII alone was demonstrated in our earlier study [4] and also by others [5, 6] . In this study, we assess whether the presence of VWF in plasma-derived FVIII preparations induces immune response against VWF in FVIII-deficient mice. Murine VWF protein has 83% identity with human pre-proVWF, and cysteine residues are conserved in both, the pro-peptide and the mature VWF sequence [7] . The FVIII preparations with different VWF content were obtained by fractionation of FVIII and the FVIII-VWF complex under identical conditions, starting with plasma-derived preparation by using size-exclusion chromatography. These preparations were administered to FVIII-deficient mice for the characterization of the antibody production against VWF and compared with mice treated with rFVIII. Plasma-derived FVIII preparations are reported to contain the cytokine transforming growth factor (TGF), which is able to suppress the proliferation of antigen-specific T cells in vitro [8] ; therefore, we also examined the impact of TGFon the development of VWF-specific antibodies. We report that the VWF antibody development differs between a mouse model and haemophilia A patients, since in haemophilia A patients, the administration of FVIII preparations containing VWF is reported not to induce VWF antibody response. Animal models have been very useful in corroborating the results obtained by in vitro studies and for the evaluation of different treatment protocols that cannot be carried through otherwise. Experiments in transgenic mice deficient in coagulation factor VIII (FVIII) as a model of haemophilia A have provided significant information. These mice develop FVIII-specific antibodies after 3–4 injections of human recombinant FVIII (rFVIII) [1] . The development of inhibitory antibodies in haemophilia A patients is the most serious consequence of treatment. Thus, it is tempting to use the FVIII-deficient mice for studying the immunogenicity of different FVIII preparations, since ex vivo models that are able to mimic this complex situation are not available so far. Von Willebrand factor (VWF), which mediates the adhesion of platelets to the subendothelium at the site of vascular injury and stabilizes FVIII by forming a noncovalent complex [2] , has been suggested to be involved in modulating the immune response to FVIII. The presence of VWF in FVIII therapeutic preparations purified from plasma increases the stability of FVIII and might be responsible for lower immunogenicity of such preparations. It has been shown that haemophilia A patients treated with VWF-containing plasma-derived FVIII preparations respond to treatment with somewhat lower production of antibodies against FVIII compared with patients treated with rFVIII preparations without VWF [3] . Employing FVIII-deficient mice, the lower producReceived: February 18, 2008 Accepted after revision: April 28, 2008 Published online: July 2, 2008