Sequential Immunization Elicits Broadly Neutralizing Anti-HIV-1 Antibodies in Ig Knockin Mice.

A vaccine that elicits broadly neutralizing antibodies (bNAbs) against HIV-1 is likely to be protective, but this has not been achieved. To explore immunization regimens that might elicit bNAbs, we produced and immunized mice expressing the predicted germline PGT121, a bNAb specific for the V3-loop and surrounding glycans on the HIV-1 spike. Priming with an epitope-modified immunogen designed to activate germline antibody-expressing B cells, followed by ELISA-guided boosting with a sequence of directional immunogens, native-like trimers with decreasing epitope modification, elicited heterologous tier-2-neutralizing responses. In contrast, repeated immunization with the priming immunogen did not. Antibody cloning confirmed elicitation of high levels of somatic mutation and tier-2-neutralizing antibodies resembling the authentic human bNAb. Our data establish that sequential immunization with specifically designed immunogens can induce high levels of somatic mutation and shepherd antibody maturation to produce bNAbs from their inferred germline precursors.

Tunable dynamics of B cell selection in gut germinal centres.

Germinal centres, the structures in which B cells evolve to produce antibodies with high affinity for various antigens, usually form transiently in lymphoid organs in response to infection or immunization. In lymphoid organs associated with the gut, however, germinal centres are chronically present. These gut-associated germinal centres can support targeted antibody responses to gut infections and immunization1. But whether B cell selection and antibody affinity maturation take place in the face of the chronic and diverse antigenic stimulation characteristic of these structures under steady state is less clear2-8. Here, by combining multicolour 'Brainbow' cell-fate mapping and sequencing of immunoglobulin genes from single cells, we find that 5-10% of gut-associated germinal centres from specific-pathogen-free (SPF) mice contain highly dominant 'winner' B cell clones at steady state, despite rapid turnover of germinal-centre B cells. Monoclonal antibodies derived from these clones show increased binding, compared with their unmutated precursors, to commensal bacteria, consistent with antigen-driven selection. The frequency of highly selected gut-associated germinal centres is markedly higher in germ-free than in SPF mice, and winner B cells in germ-free germinal centres are enriched in 'public' clonotypes found in multiple individuals, indicating strong selection of B cell antigen receptors even in the absence of microbiota. Colonization of germ-free mice with a defined microbial consortium (Oligo-MM12) does not eliminate germ-free-associated clonotypes, yet does induce a concomitant commensal-specific B cell response with the hallmarks of antigen-driven selection. Thus, positive selection of B cells can take place in steady-state gut-associated germinal centres, at a rate that is tunable over a wide range by the presence and composition of the microbiota.

Effects of factor VIII levels on the APTT and anti-Xa activity under a therapeutic dose of heparin.

In pregnant women, activated partial thromboplastin time (APTT) does not precisely reflect the anticoagulant effect of a therapeutic dose of heparin. However, the measurement of anti-Xa activity can be used to monitor the anticoagulant effect of heparin, since the plasma concentrations of coagulation factors increase in pregnant women. We evaluated the in vitro effects of increased concentrations of fibrinogen and other coagulation factors (FVII, FVIII, and FIX) on the results of assays of APTT and anti-Xa activity in plasma samples with various therapeutic concentrations of unfractionated heparin (UFH). In the presence of UFH, APTT was shortened by increased concentrations of fibrinogen, FVII, or FVIII, and this effect was much stronger when the FVIII concentration was increased. In the plasma samples containing 0.5 or 0.7 U/mL of UFH, the APTT was shortened by approximately half or one-third, respectively, when 6 U FVIII/mL was added to the sample. The anti-Xa activity was not influenced by increased concentrations of the coagulation factors. In the present study, we also evaluated the sensitivities to UHF of four APTT reagents, and found a 1.65-fold difference in the sensitivity to UFH among APTT reagents. Our results demonstrate that increased FVIII concentration shortens APTT under therapeutic doses of UFH, and that APTT thus underestimates the anticoagulant effect of UFH in pregnant women, mainly due to the increased FVIII concentration.

The tumor suppressor Smad4/DPC4 is regulated by phosphorylations that integrate FGF, Wnt, and TGF-ß signaling.

Smad4 is a major tumor suppressor currently thought to function constitutively in the transforming growth factor ß (TGF-ß)-signaling pathway. Here, we report that Smad4 activity is directly regulated by the Wnt and fibroblast growth factor (FGF) pathways through GSK3 and mitogen-activated protein kinase (MAPK) phosphorylation sites. FGF activates MAPK, which primes three sequential GSK3 phosphorylations that generate a Wnt-regulated phosphodegron bound by the ubiquitin E3 ligase ß-TrCP. In the presence of FGF, Wnt potentiates TGF-ß signaling by preventing Smad4 GSK3 phosphorylations that inhibit a transcriptional activation domain located in the linker region. When MAPK is not activated, the Wnt and TGF-ß signaling pathways remain insulated from each other. In Xenopus embryos, these Smad4 phosphorylations regulate germ-layer specification and Spemann organizer formation. The results show that three major signaling pathways critical in development and cancer are integrated at the level of Smad4.

Combining FVIIa and FX into a mixture which imparts a unique thrombin generation potential to hemophilic plasma: an in vitro assessment of FVIIa/FX mixture as an alternative bypassing agent.

INTRODUCTION: We previously reported that a combination of factors VIIa (FVIIa) and X (FX) might represent an effective and attractive alternative to recombinant factor VIIa (rFVIIa) and plasma-derived activated prothrombin complex concentrate (APCC) for controlling bleeding in hemophiliacs with inhibitors. The present study describes the standardization and preparation of a virus-inactivated and nano-filtrated plasma-derived FVIIa/FX concentrate. We hypothesized that the hemostatic capacity was equivalent to or better than current bypassing agents as evaluated by measurements of waveform APTT clotting and thrombin generation. RESULTS: Kinetic analyses showed that a "normal" FX concentration of approximately 140nM in plasma did not induce maximum catalytic efficacy of FVIIa and that an increase in the concentration of FX in hemophilic plasma enhanced the thrombin generation potential of FVIIa. Thus, the FVIIa/FX mixture was prepared by assembling plasma-derived FVIIa and FX at a weight ratio of 1:10. The FVIIa/FX mixture proved superior to rFVIIa with regards to shortening the APTT and accelerating the thrombin generation in hemophilic plasma. The FVIIa/FX mixture promoted the generation of thrombin more than did rFVIIa. CONCLUSIONS: Increasing the FX concentration in hemophilic plasma gives a higher clotting potential of FVIIa. A FVIIa/FX concentrate may serve as a new alternative bypassing agent.

Von Willebrand factor accelerates platelet adhesion and thrombus formation on a collagen surface in platelet-reduced blood under flow conditions.

Plasma von Willebrand factor (VWF) has been identified as an indispensable factor for platelet adhesion and thrombus formation on a collagen surface under flow conditions. VWF binds to collagen and then tethers platelets to the collagen surface through interaction with platelet glycoprotein Ib and also contributes to the thrombus formation on the collagen surface. In the present study, we demonstrated that the addition of VWF/factor VIII complex or purified VWF (> 2 ristocetin cofactor activity units/mL) increased platelet adhesion to the collagen surface in platelet-reduced blood ( approximately 5 x 10(4) platelets/microL) to the normal level. VWF had no stimulatory effect when it was allowed to bind to the collagen surface before blood flow was initiated. Addition of an excess of FITC (fluorescein-5-isothiocyanate)-labeled VWF to platelet-reduced blood under these flow conditions demonstrated that the VWF was mainly incorporated into the platelet aggregates. These results indicated that the supplemented VWF stimulates the platelet adhesion onto the collagen surface by enhancing platelet aggregation in the platelet-reduced condition. This also suggests a possibility that supplementation of VWF to individuals with thrombocytopenia might be effective for increasing their hemostatic potential.