Memory B cells are responsible for the rapidly emerging antibody response after antigen reexposure. The signals required for the restimulation of memory B cells have not been fully explained. We used a murine model of anti-factor VIII (FVIII) antibody responses in hemophilia A to study the requirements for the restimula-tion of FVIII-specific memory B cells and their differentiation into anti-FVIII anti-body-producing cells. We were particularly interested in the significance of activated T cells and costimulatory interactions. Our results indicate that the re-stimulation of FVIII-specific memory B cells is strictly dependent on interactions with activated T cells. These activated T cells can be specific for either FVIII or third-party antigens. Restimulation by T cells specific for third-party antigens requires the presence of FVIII, indicating that signals induced by B-cell receptor (BCR) triggering and by interactions with activated T cells are important. The blockade of B7-1 or B7-2 as well as the blockade of CD40L inhibits the restimulation and differentiation of FVIII-specific memory B cells in vitro and in vivo. The interference with inducible costimulator-inducible costimulator ligand (ICOS-ICOSL) interactions, however, does not cause any modulation. As expected, the production of anti-FVIII antibodies by plasma cells is not dependent on any of the costimulatory interactions tested.

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...

Bloodstream infections (BSI) and sepsis are major causes of morbidity and mortality worldwide. Blood culture-based diagnostics usually requires 1-2 days for identification of bacterial agent and an additional 2-3 days for phenotypic determination of antibiotic susceptibility pattern. With the escalating burden of antimicrobial resistance (AMR) rapid diagnostics becomes increasingly important to secure adequate antibiotic therapy. Real-time whole genome sequencing represents a genotypic diagnostic approach with the ability to rapidly identify pathogens and AMR-encoding genes. Here we have used nanopore sequencing of bacterial DNA extracted from positive blood cultures for identification of pathogens, detection of plasmids and AMR-encoding genes. To our knowledge, this is the first study to gather the above-mentioned information from nanopore sequencing and conduct a comprehensive analysis for diagnostic purposes in real-time. Identification of pathogens was possible after 10 minutes of sequencing and all predefined AMR-encoding genes and plasmids from monoculture experiments were detected within one hour using raw nanopore sequencing data. Furthermore, we demonstrate the correct identification of plasmids and bla CTX-M subtypes using de novo assembled nanopore contigs. Results from this study hold great promise for future applications in clinical microbiology and for health care surveillance purposes. Bloodstream infections (BSIs) and sepsis are major causes of morbidity and mortality worldwide. Epidemiological data are scarce, but a recent estimate indicated that 31.5 million cases of sepsis and 5.3 million sepsis attributable deaths occur annually 1. This estimate is only based on data collected from high-income countries, and it therefore likely underestimates the true burden of disease worldwide, especially in low-and-middle-income countries 2. Most studies on sepsis and BSIs report an increasing incidence over the last two decades 3 , particularly among the immunocompromised, multimorbid, and elderly patients, or due to failure of empiric antibiotic regimens as result of antimicrobial resistance (AMR) 4. With multi drug resistant pathogens spreading at an alarming rate, widely adopted empirical antibiotic treatment regimens for sepsis based on penicillin (or aminopenicillin) in combination with gentamicin 5 are being challenged. In particular, the escalating burden of infections due to extended-spectrum β-lactamase (ESBL) producing Gram negative bacteria represents a major health concern. These bacteria, mainly Escherichia coli and Klebsiella pneumoniae, are not only resistant to all penicillins and third generation cephalosporins, but also frequently express co-resistance to gentamicin. Consequently, treatment failure may occur, and clinicians increasingly prescribe last-resort antibiotics such as carbapenems as initial antibiotic treatment of sepsis. This in turn contributes to development and spread of AMR and to a further increase in the burden of infections caused by resistant bacteria. Current state-of-...