Factor VIII trafficking to CD4+ T cells shapes its immunogenicity and requires several types of antigen-presenting cells.

Despite >80 years of clinical experience with coagulation factor VIII (FVIII) inhibitors, surprisingly little is known about the in vivo mechanism of this most serious complication of replacement therapy for hemophilia A. These neutralizing antidrug alloantibodies arise in ∼30% of patients. Inhibitor formation is T-cell dependent, but events leading up to helper T-cell activation have been elusive because of, in part, the complex anatomy and cellular makeup of the spleen. Here, we show that FVIII antigen presentation to CD4+ T cells critically depends on a select set of several anatomically distinct antigen-presenting cells, whereby marginal zone B cells and marginal zone and marginal metallophilic macrophages but not red pulp macrophages (RPMFs) participate in shuttling FVIII to the white pulp in which conventional dendritic cells (DCs) prime helper T cells, which then differentiate into follicular helper T (Tfh) cells. Toll-like receptor 9 stimulation accelerated Tfh cell responses and germinal center and inhibitor formation, whereas systemic administration of FVIII alone in hemophilia A mice increased frequencies of monocyte-derived and plasmacytoid DCs. Moreover, FVIII enhanced T-cell proliferation to another protein antigen (ovalbumin), and inflammatory signaling-deficient mice were less likely to develop inhibitors, indicating that FVIII may have intrinsic immunostimulatory properties. Ovalbumin, which, unlike FVIII, is absorbed into the RPMF compartment, fails to elicit T-cell proliferative and antibody responses when administered at the same dose as FVIII. Altogether, we propose that an antigen trafficking pattern that results in efficient in vivo delivery to DCs and inflammatory signaling, shape the immunogenicity of FVIII.

KIF3A accelerates KIF3C within the kinesin-2 heterodimer to generate symmetrical phosphate release rates for each processive step.

Heterodimeric KIF3AC is a mammalian kinesin-2 that is highly expressed in the central nervous system and associated with vesicles in neurons. KIF3AC is an intriguing member of the kinesin-2 family because the intrinsic kinetics of KIF3A and KIF3C when expressed as homodimers and analyzed in vitro are distinctively different from each other. For example, the single-molecule velocities of the engineered homodimers KIF3AA and KIF3CC are 293 and 7.5 nm/s, respectively, whereas KIF3AC has a velocity of 186 nm/s. These results led us to hypothesize that heterodimerization alters the intrinsic catalytic properties of the two heads, and an earlier computational analysis predicted that processive steps would alternate between a fast step for KIF3A followed by a slow step for KIF3C resulting in asymmetric stepping. To test this hypothesis directly, we measured the presteady-state kinetics of phosphate release for KIF3AC, KIF3AA, and KIF3CC followed by computational modeling of the KIF3AC phosphate release transients. The results reveal that KIF3A and KIF3C retain their intrinsic ATP-binding and hydrolysis kinetics. Yet within KIF3AC, KIF3A activates the rate of phosphate release for KIF3C such that the coupled steps of phosphate release and dissociation from the microtubule become more similar for KIF3A and KIF3C. These coupled steps are the rate-limiting transition for the ATPase cycle suggesting that within KIF3AC, the stepping kinetics are similar for each head during the processive run. Future work will be directed to define how these properties enable KIF3AC to achieve its physiological functions.

Kinesin-2 heterodimerization alters entry into a processive run along the microtubule but not stepping within the run.

Heterodimeric KIF3AC and KIF3AB, two members of the mammalian kinesin-2 family, generate force for microtubule plus end-directed cargo transport. However, the advantage of heterodimeric kinesins over homodimeric ones is not well-understood. We showed previously that microtubule association for entry into a processive run was similar in rate for KIF3AC and KIF3AB at ∼7.0 µm-1 s-1 Yet, for engineered homodimers of KIF3AA and KIF3BB, this constant is significantly faster at 11 µm-1 s-1 and much slower for KIF3CC at 2.1 µm-1 s-1 These results led us to hypothesize that heterodimerization of KIF3A with KIF3C and KIF3A with KIF3B altered the intrinsic catalytic properties of each motor domain. Here, we tested this hypothesis by using presteady-state stopped-flow kinetics and mathematical modeling. Surprisingly, the modeling revealed that the catalytic properties of KIF3A and KIF3B/C were altered upon microtubule binding, yet each motor domain retained its relative intrinsic kinetics for ADP release and subsequent ATP binding and the nucleotide-promoted transitions thereafter. These results are consistent with the interpretation that for KIF3AB, each motor head is catalytically similar and therefore each step is approximately equivalent. In contrast, for KIF3AC the results predict that the processive steps will alternate between a fast step for KIF3A followed by a slow step for KIF3C resulting in asymmetric stepping during a processive run. This study reveals the impact of heterodimerization of the motor polypeptides for microtubule association to start the processive run and the fundamental differences in the motile properties of KIF3C compared with KIF3A and KIF3B.

Do Anesthesia Residents perceive a Benefit from participating in Bedside Tracheostomies?

BACKGROUND: Airway management is a core competency in anesthesiology training and practice. Residents are taught how to evaluate patients and identify those who may difficulty in securing their airway. The ASA has devised an algorithm on management of those difficult airways. The conservative methods are taught and practiced throughout training. However, the default last resort is obtaining an invasive airway. It is this potentially life-saving, procedure that residents may graduate and have never performed clinically. In our institution, the Anesthesiology Critical Care Division routinely performs percutaneous tracheostomies throughout the hospital. As residents began to inquire, they too were folded into this service to provide real hands on experience. After 3 years we sought to determine if residents perceived this hands-on training to be a benefit. METHODS: We devised a multi-question survey and distributed to our 131 residents. The purpose of the survey was to determine of those who participated in the tracheostomy service if they felt this was of benefit, which specialists they could look to should an invasive airway be needed and if they felt this exposure gave them greater confidence to perform an emergency invasive airway. RESULTS: In unanimity, the residents all felt that this training was both beneficial and essential in their training. However, none of the residents felt this training had adequately prepared them to actually perform this procedure in an emergency. CONCLUSIONS: The residents felt this was an essential aspect of their anesthesiology training. However they did not feel they obtained invasive airway competence. We postulate the residents relatively limited exposure may have been the cause. While we do not know the impact of this training in residency on management of a future difficult airway, the residents uniformly felt this was vital in their clinical curriculum and should be universal.