Rouzbeh Chegeni

Anti-CD3 prevents factor VIII inhibitor development in hemophilia A mice by a regulatory CD4+CD25+-dependent mechanism and by shifting cytokine production to favor a Th1 response

Non-Fc-receptor binding anti-CD3 Ab therapy, in the setting of several different autoimmune disorders, can induce antigen-specific and long-lasting immunologic tolerance. Because factor VIII (FVIII) inhibitor formation is the most serious treatment-related complication for hemophilia A patients, we tested the efficacy of anti-CD3 to prevent FVIII inhibitor formation in hemophilia A BALB/c and C57BL/6 mice. A short course of low-dose anti-CD3 significantly increased expression of CD25 and the proportion of CD4+CD25+ regulatory T cells in the spleen and potently prevented the production of inhibitory and non-neutralizing anti-FVIII antibodies in both strains of mouse. Depleting the CD4+CD25+ cells during anti-CD3 therapy completely ablated tolerance to FVIII. Further phenotypic characterization of regulatory cells in tolerant mice showed a consistently higher number of CD4+GITR+ and CD4+FoxP3+ cells in both strains of mice. In addition, in tolerant C57BL/6 mice we observed an increase in CD4+CD25+ CTLA-4+ and CD4+CD25+mTGF-beta1+ cells. Finally, in vitro cytokine profiling demonstrated that splenocytes from tolerant BALB/c and C57BL/6 were polarized toward a Th1-immune response. Taken together, these findings indicate that anti-CD3 induces tolerance to FVIII and that the mechanism(s) regulating this response almost certainly occurs through the generation of several distinct regulatory T-cell lineages and by influencing cytokine production and profile.

Recombinant and plasma-derived factor VIII products induce distinct splenic cytokine microenvironments in hemophilia A mice

The use of plasma-derived factor VIII (pdFVIII) concentrates in hemophilia A has been reported to result in reduced anti-FVIII antibody formation. In this study, we have investigated whether the cytokine microenvironment induced by pdFVIII has an influence on reducing anti-FVIII antibody titers in hemophilic mice. Microarray and confirmatory quantitative reverse transcription polymerase chain reaction (RT-PCR) experiments show that pdFVIII infusion causes a different transcriptional profile in dendritic cells than recombinant FVIII (rFVIII). Both treatments caused up-regulation of proinflammatory gene expression, but rFVIII and pdFVIII treatments promote expression of genes that induce Th1 and Th2 responses, respectively. Moreover, administration of rFVIII or pdFVIII concentrates resulted in distinct T-cell splenic cytokine microenvironments. rFVIII induced the release of Th1 cytokines and IL-10, whereas pdFVIII induced the release of Th2 cytokines and transforming growth factor-beta. We have also observed high titers of anti-human von Willebrand factor (VWF) antibodies in the pdFVIII-treated mice and propose that this results from antigenic competition. We further investigated the role of this phenomenon using infusions of FVIII and increasing concentrations of recombinant human factor IX (FIX). These studies show an inverse relationship between increasing concentrations of FIX and the production of anti-FVIII antibodies. In summary, these studies report new mechanisms that contribute to reduced anti-FVIII antibody development in hemophilia A after pdFVIII infusions.

Reduction of the immune response to factor VIII mediated through tolerogenic factor VIII presentation by immature dendritic cells

Summary.  Background: The development of neutralizing antibodies to factor FVIII (FVIII) represents the most serious complication in the treatment of hemophilia A. Objective: We have explored the potential of using immature dendritic cells (iDCs) to present FVIII in a tolerogenic manner to T cells. Methods: The iDCs were isolated from hemophilic murine bone marrow and pulsed with canine cFVIII (cFVIII‐iDCs) in the presence or absence of the NFκB pathway blocking compound Andrographolide (Andro‐cFVIII‐iDCs). Three weekly intravenous infusions of one million cFVIII pulsed‐iDCs were administered to a group of five hemophilic Balb/c mice. Anti‐FVIII antibody levels were monitored by functional Bethesda assay after four weekly intravenous challenges with 2 IU of cFVIII. Results: We have shown that cFVIII in the presence or absence of Andro is efficiently taken up by iDCs and that this process does not result in the maturation of DCs or the activation of co‐cultured T cells. Following repeated infusion of the cFVIII‐iDCs and Andro‐cFVIII‐iDCs into hemophilic mice, which were subsequently challenged with cFVIII, long‐term reductions of FVIII inhibitors of 25% and 40%, respectively, were documented. Studies of cytokine release and T‐cell phenotypes indicate that the mechanisms responsible for reducing immunologic responsiveness to cFVIII appear to involve an expansion of Foxp3 T regulatory cells in the case of cFVIII‐iDC infusion and the elaboration of the immunosuppressive cytokines IL‐10 and TGF‐β following andrographolide‐treated cFVIII‐iDCs. Conclusions: This study shows that tolerogenic presentation of cFVIII to the immune system can significantly reduce immunogenicity of the protein.

Immunoglobulin isotypes and functional anti-FVIII antibodies in response to FVIII treatment in Balb/c and C57BL/6 haemophilia A mice

Summary.  Previous studies have demonstrated that genetic factors play an important role in determining the likelihood of formation of anti‐factor VIII (FVIII) antibodies in haemophilia A patients. We were interested in characterizing the spectrum of FVIII antibody formation and the primary and secondary immune responses after FVIII administration in two different exon 16‐disrupted haemophilia A mouse strains, Balb/c and C57BL/6. Balb/c and C57BL/6 E16 haemophilia A mice were used in all experiments. Total FVIII antibodies and FVIII inhibitors were measured using ELISA and Bethesda assays respectively. T‐ and B‐cell cytokines were quantified using ELISA and flow cytometry. FVIII antibodies, but not functional inhibitors were detectable 1 week after the first FVIII treatment in both strains. These antibodies mainly belonged to the IgM and IgA isotypes. After the fourth FVIII treatment, neutralizing anti‐FVIII antibodies were detected in both mouse strains: Balb/c (mean inhibitory titer 58 BU) and C57BL/6 (mean inhibitory titer 82 BU). IgG1 levels were similar in both strains but the IgG2A and IgG2B subclasses were higher in C57BL/6 mice. The results of intracellular cytokine staining of T cells indicated that the FVIII‐treated C57BL/6 mice produced more IL10 and Th1 cytokines than the FVIII‐treated Balb/c mice. These studies show that C57BL/6 mice develop a stronger immune response towards FVIII than Balb/c mice. We propose that the enhanced Th1 and IL10 cytokine micro‐environment induced in C57BL/6 mice is responsible for this difference. Therefore, genetic strain‐dependent differences must be considered when evaluating immunological outcomes in mouse models of haemophilia A.

Evaluation of H3 histone methylation and colony formation in erythroid progenitors treated with thalidomide and sodium butyrate.

OBJECTIVES β-thalassemia and sickle cell disease are hemoglobinopathies with reduced/absent β chains in the former and dysfunctional β chains in the latter. In both conditions, up-regulation of hemoglobin F through demethylation can alleviate the symptoms. This can be attained with drugs such as thalidomide and sodium butyrate. MATERIALS AND METHODS This study was performed on erythroid progenitors derived from CD133+ cord blood stem cells. Erythroid progenitors were treated with thalidomide and sodium butyrate in single and combined groups. Colony-formation potential in each group was evaluated by the colony assay. Real-time polymerase chain reaction (RT-PCR) was used to evaluate the effect of these drugs on histone H3 lysine 27 (H3K27) methylation patterns. FINDINGS Compared to other treatment groups, CD133+ cells treated with thalidomide alone produced more hematopoietic colonies. Thalidomide alone was also more effective in decreasing H3K27 methylation. CONCLUSIONS Thalidomide shows superiority to sodium butyrate as a hypomethylating agent in this cell culture study, and it has the potential to become conventional treatment for sickle cell disease and β-thalassemia.

Functional analysis of three recombinant A1-VWF domain mutants in comparison to wild type and plasma-derived VWF facilitates subtyping in type 2 von Willebrand disease

Phenotypic diagnosis of VWD, in particular type 2, is challenging. Molecular diagnosis may fail to provide clarity since mutations within a short stretch of the same domain may cause various phenotypes, and since even experts will ascribe different subtypes to similar mutations. We assessed diagnostic difficulty in VWD by investigating five cases where phenotypic data was unclear. We identified 3 novel mutations within the A1 domain of the VWF gene: L1460F (2 related patients), Y1363C (1 patient), E1389K (2 related patients). These were not found in 100 normal individuals or documented in the VWF mutation database. Detailed functional analysis of recombinant mutants included VWF multimers, VWF:Ag, VWF:RCo, VWF:CB, and Platelet-VWF binding studies, and results assessed against recombinant WT and plasma derived (pd) VWF. Multimer analysis showed clear loss of HMW VWF with E1389K only, consistent with coincident low relative CB/Ag ratio. VWF-platelet binding studies using two independent approaches showed enhanced activity for L1460F, but reduced activity for E1389K and Y1363C. A novel finding was that WT rVWF showed enhanced platelet binding in RIPA analysis compared to pdVWF with this being dependent on the dilution material used. Through these extensive studies, we assigned L1460F to type 2B, E1389K to 2A, and Y1363C to 2M VWD. Thus, although molecular analysis is not required to classify VWD patient subtypes, a thorough and combined phenotypic, genotypic and functional analysis will assist assignment of the VWD subtype.

Corrigendum to “Factor V Mutations in Iranian patients with activated protein C resistance and venous thrombosis” [Thrombosis Research 119/2 (2007) 189–193]

a Department of Hematology and Blood Banking, School of Medicine, Tarbiat Modarres University, Jalale-ale-Ahmad Ave., Tehran, Iran b Cellular and Molecular Biology Research Center, Shaheed Beheshti University of Medical Sciences, Tehran, Iran c Department of Hematology, College of Medicine, Shaheed Beheshti University of Medical Sciences, Tehran, Iran d Comprehensive Hemophilia Center of Iran, Iran