IntroductionThe development of neutralizing anti-factor VIII (FVIII) antibodies is the major complication in the treatment of patients with hemophilia A with FVIII products. 1,2 Long-term application of high doses of FVIII has evolved as an effective therapy to eradicate the antibodies and to induce long-lasting immune tolerance. [3][4][5][6] Despite clinical experience with the therapy, little is known about the immunologic mechanisms that cause the down-modulation of FVIII-specific immune responses and the induction of long-lasting immune tolerance against FVIII. We asked the question whether the restimulation of FVIII-specific memory B cells is affected by high concentrations of FVIII in vitro or high doses of FVIII in vivo. Memory B cells play an essential role in the maintenance of established antibody responses. On re-exposure to the same antigen, they are rapidly restimulated to proliferate and differentiate into antibody-secreting plasma cells (ASCs) that secrete high-affinity antibodies. 7,8 Furthermore, memory B cells have the potential to act as very efficient antigen-presenting cells and stimulators of CD4 ϩ T cells because of the expression of highaffinity antigen receptors, major histocompatibility complex (MHC) class II and costimulatory molecules. 9 It is, therefore, reasonable to believe that memory B cells have to be eradicated or functionally inactivated during a successful immune tolerance induction therapy with FVIII inhibitors in patients with hemophilia A.We used a murine model of hemophilia A that is characterized by complete deficiency of functionally active FVIII because of a targeted disruption of exon 17 of the F8 gene. 10,11 Intravenous injection of human FVIII into these mice results in high titers of anti-FVIII antibodies that have similar characteristics to those of FVIII inhibitors in patients. [12][13][14][15] Using this model, we demonstrated previously that the differentiation of FVIII-specific memory B cells into ASCs depends on the presence of activated T cells and requires CD40-CD40 ligand and CD80/CD86-CD28 costimulatory interactions. 16 Here, we show that concentrations of FVIII below the physiologic plasma concentration of 0.1 g/mL (1 U/mL) restimulate FVIII-specific memory B cells and induce their differentiation into ASCs. Concentrations above 0.1 g/mL (1 U/mL), however, inhibit memory B-cell restimulation and prevent the formation of ASCs. This inhibition is irreversible and involves the activation of caspases. Materials and methods Hemophilic E-17 miceOur colony of fully inbred hemophilic E-17 mice (characterized by a targeted disruption of exon 17 of the F8 gene) was established with a breeding pair from the original colony 10,11 and crossed into the C57BL/6J background as described. 17 All mice were male and aged 8 to 10 weeks at the beginning of the experiments. All studies were carried out in accordance with Austrian federal law (Act BG 501/1989) regulating animal experimentation and approved by the local authority in Vienna, Austria. Immunization of mice with FV...
In human immunodeficiency virus (HIV) infection, persistent inflammation despite effective antiretroviral therapy (ART) is linked to increased risk of non-infectious chronic complications such as cardiovascular and thromboembolic disease. A better understanding of inflammatory and coagulation pathways in HIV infection is needed to optimize clinical care. Markers of monocyte activation and coagulation independently predict morbidity and mortality associated with non-AIDS events. In this study, we identified a specific subset of monocytes that express tissue factor (TF), persist after virological suppression and trigger the coagulation cascade by activating factor X. This subset of monocytes expressing TF had a distinct gene signature with upregulated innate immune markers as well as evidence of robust production of multiple proinflammatory cytokines including IL-1β, TNF-α, and IL-6 ex vivo and in vitro upon LPS stimulation. We validated our findings in a nonhuman primate model, showing that TF-expressing inflammatory monocytes were associated with SIV-related coagulopathy in the progressive (pigtail macaques) but not the non-pathogenic (African Green Monkeys) SIV infection model. Lastly, Ixolaris, an anti-coagulant that inhibits the TF pathway, was tested and potently blocked functional TF activity in vitro in HIV and SIV infection without affecting monocyte responses to toll-like receptor (TLR) stimulation. Strikingly, in vivo treatment of chronically infected PTMs with Ixolaris was associated with significant decreases in D-dimer and immune activation. These data suggest that TF expressing monocytes are at the epicenter of inflammation and coagulation in chronic HIV and SIV infection and may represent a potential therapeutic target.
Although there have been numerous studies on the fate of injected iron compounds in experimental animals (1-8), the transformation of the iron compounds within individual cells has not yet been traced at the molecular level. Recent work in which electron microscopy was combined with other techniques (9-12) has provided information on the molecular structure and the disposition of hemosiderin in several types of cells under varying circumstances, on the relationship of hemosiderin to ferritin, and on the possible role of specialized mitochondria ("siderosomes") in hemosiderosis. In the work now to be presented it has proved possible to distinguish intracellular deposits of iron compounds, given parenterally, from ferritin and from indigenous hemosiderin, and to gain insight into the transformation of the injected iron compounds into ferritin and hemosiderin. The findings bear on several aspects of iron metabolism, especially on the utilization of colloidal iron preparations within macrophages and endothelial cells. General Plan of ExperimentsThe general plan of the experiments was, first, to trace the sequence of changes in the fine structure of intracellular deposits of injected iron compounds in several types of cells in mice and rats at various intervals following intraperitoneal injection; second, to compare the physico-chemical nature of the intracellular material with that of the iron compounds prior to their injection; tkirdly, to confirm the actual presence of ferritin, about or in relation to deposits
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