CD4+ T cell calibration of antigen-presenting cells optimizes antiviral CD8+ T cell immunity.

Antiviral CD8+ T cell immunity depends on the integration of various contextual cues, but how antigen-presenting cells (APCs) consolidate these signals for decoding by T cells remains unclear. Here, we describe gradual interferon-α/interferon-ß (IFNα/ß)-induced transcriptional adaptations that endow APCs with the capacity to rapidly activate the transcriptional regulators p65, IRF1 and FOS after CD4+ T cell-mediated CD40 stimulation. While these responses operate through broadly used signaling components, they induce a unique set of co-stimulatory molecules and soluble mediators that cannot be elicited by IFNα/ß or CD40 alone. These responses are critical for the acquisition of antiviral CD8+ T cell effector function, and their activity in APCs from individuals infected with severe acute respiratory syndrome coronavirus 2 correlates with milder disease. These observations uncover a sequential integration process whereby APCs rely on CD4+ T cells to select the innate circuits that guide antiviral CD8+ T cell responses.

Terpene biosynthesis in marine sponge animals.

Sea sponges are the largest marine source of small-molecule natural products described to date. Sponge-derived molecules, such as the chemotherapeutic eribulin, the calcium-channel blocker manoalide, and antimalarial compound kalihinol A, are renowned for their impressive medicinal, chemical, and biological properties. Sponges contain microbiomes that control the production of many natural products isolated from these marine invertebrates. In fact, all genomic studies to date investigating the metabolic origins of sponge-derived small molecules concluded that microbes-not the sponge animal host-are the biosynthetic producers. However, early cell-sorting studies suggested the sponge animal host may play a role particularly in the production of terpenoid molecules. To investigate the genetic underpinnings of sponge terpenoid biosynthesis, we sequenced the metagenome and transcriptome of an isonitrile sesquiterpenoid-containing sponge of the order Bubarida. Using bioinformatic searches and biochemical validation, we identified a group of type I terpene synthases (TSs) from this sponge and multiple other species, the first of this enzyme class characterized from the sponge holobiome. The Bubarida TS-associated contigs consist of intron-containing genes homologous to sponge genes and feature GC percentage and coverage consistent with other eukaryotic sequences. We identified and characterized TS homologs from five different sponge species isolated from geographically distant locations, thereby suggesting a broad distribution amongst sponges. This work sheds light on the role of sponges in secondary metabolite production and speaks to the possibility that other sponge-specific molecules originate from the animal host.

Constitutive Flt3 signaling impacts conventional dendritic cell function.

The development of dendritic cells (DCs) depends on signaling via the FMS-like tyrosine kinase 3 (Flt3) receptor. How Flt3 signaling impacts terminally differentiated DC function is unknown. This is important given the increasing interest in exploiting Flt3 for vaccination and tumor immunotherapy. Here, we examined DCs in mice harboring constitutively activated Flt3 (Flt3-ITD). Flt3ITD/ITD mice possessed expanded splenic DC subsets including plasmacytoid DC, conventional DC (cDC)1, cDC2, double positive (DP) cDC1 (CD11c+ CD8+ CD11b- CD103+ CD86+), noncanonical (NC) cDC1 (CD11c+ CD8+ CD11b- CD103- CD86-) and single positive (SP) cDC1 (CD11c+ CD8+ CD11b- CD103- CD86+). Outcomes of constitutive Flt3 signaling differed depending on the cDC subset examined. In comparison with wild type (WT) DCs, all Flt3ITD/ITD cDCs displayed an altered surface phenotype with changes in costimulatory molecules, major histocompatibility complex class I (MHC I) and II (MHC II). Cytokine secretion patterns, antigen uptake, antigen proteolysis and antigen presenting function differed between WT and Flt3ITD/ITD subsets, particularly cDC2. In summary, Flt3 signaling impacts the function of terminally differentiated cDCs with important consequences for antigen presentation.

Generalization of savoring to novel positive stimuli.

Savoring is a positive emotion up-regulation technique that can increase electrocortical and self-reported valence and arousal to positive and neutral pictures, with effects persisting to increase response to the same stimuli when encountered later. Outside of the lab, emotion regulation techniques that persist to affect not just encounters with the same stimuli but also encounters with similar, but previously unencountered stimuli should save individuals time and effort. Here, we used event-related potentials and picture ratings to test whether savoring would generalize to similar, but previously unseen positive pictures. To this end, 89 participants (56 female; M age = 18.96 years, SD = 1.87) were asked to savor positive pictures from one category (e.g., happy people) and to view positive pictures from another category (e.g., cute animals), as well as to view neutral pictures (e.g., plants). In a subsequent passive picture viewing task, participants viewed novel pictures from all three categories (i.e., happy people, cute animals, plants). In the first task, savoring was effective for pictures of animals throughout picture presentation, but only for pictures of people during the later part of picture presentation. In the second task, savoring generalized to novel pictures of animals, though this was only evident in the early portion of picture processing (and for self-reported ratings). Therefore, savoring holds promise as a useful technique for increasing positive emotion in everyday life, though more work is needed to understand whether effects may vary depending on different types of picture content.

Comparative safety and efficacy of a hybrid intravenous and oral diltiazem protocol for acute rate control in the emergency department.

STUDY OBJECTIVE: Intravenous diltiazem has experienced numerous supply shortages over the past few years. The purpose of this study was to compare the safety and efficacy of a traditional diltiazem intravenous bolus and continuous infusion protocol to a diltiazem intravenous bolus and oral maintenance protocol for acute rate control in the emergency department. METHODS: Patients who received intravenous diltiazem in the emergency department between January 1, 2018 and May 31, 2019 were screened. Patients were included if they received the diltiazem intravenous bolus and continuous infusion protocol (IV + infusion group) or the hybrid diltiazem intravenous bolus and oral maintenance protocol (IV + PO group). The primary outcome was the proportion of patients with rate control, without need for additional rate control agents or additional boluses during the maintenance phase. RESULTS: A total of 106 patients were matched with 53 patients in each group. For the primary outcome of rate control at four hours, 62.3% of patients in the intravenous bolus + infusion group versus 75.5% of patients in the IV bolus + PO group (p = 0.142) achieved rate control. There was no difference in rates of hypotension or bradycardia between groups. CONCLUSION: Results of this study demonstrated no difference in acute rate control when using a hybrid IV and oral diltiazem protocol, compared to a traditional IV bolus and infusion strategy. This information supports the further use of a hybrid diltiazem IV and oral protocol, which provides increased flexibility during shortages of either medication.

Identification of Isonitrile-Containing Natural Products in Complex Biological Matrices through Ligation with Chlorooximes.

Isonitrile-containing natural products have garnered attention for their manifold bioactivities but are difficult to detect and isolate due to the chemical lability of the isonitrile functional group. Here, we used the isonitrile-chlorooxime ligation (INC) in a reactivity-based screening (RBS) protocol for the detection and isolation of alkaloid and terpene isonitriles in the cyanobacterium Fischerella ambigua and a marine sponge of the order Bubarida, respectively. A trifunctional probe bearing a chlorooxime moiety, a UV active aromatic moiety, and a bromine label facilitated the chemoselective reaction with isonitriles, UV-Vis spectroscopic detection, and mass spectrometric analysis. The INC-based RBS allowed for the detection, isolation, and structural elucidation of isonitriles in microgram quantities.

MHC Class II Ubiquitination Regulates Dendritic Cell Function and Immunity.

MHC class II (MHC II) Ag presentation by dendritic cells (DCs) is critical for CD4+ T cell immunity. Cell surface levels of MHC II loaded with peptide is controlled by ubiquitination. In this study, we have examined how MHC II ubiquitination impacts immunity using MHC IIKRKI/KI mice expressing mutant MHC II molecules that are unable to be ubiquitinated. Numbers of conventional DC (cDC) 1, cDC2 and plasmacytoid DCs were significantly reduced in MHC IIKRKI/KI spleen, with the remaining MHC IIKRKI/KI DCs expressing an altered surface phenotype. Whereas Ag uptake, endosomal pH, and cathepsin protease activity were unaltered, MHC IIKRKI/KI cDC1 produced increased inflammatory cytokines and possessed defects in Ag proteolysis. Immunization of MHC IIKRKI/KI mice identified impairments in MHC II and MHC class I presentation of soluble, cell-associated and/or DC-targeted OVA via mAb specific for DC surface receptor Clec9A (anti-Clec9A-OVA mAb). Reduced T cell responses and impaired CTL killing was observed in MHC IIKRKI/KI mice following immunization with cell-associated and anti-Clec9A-OVA. Immunization of MHC IIKRKI/KI mice failed to elicit follicular Th cell responses and generated barely detectable Ab to anti-Clec9A mAb-targeted Ag. In summary, MHC II ubiquitination in DCs impacts the homeostasis, phenotype, cytokine production, and Ag proteolysis by DCs with consequences for Ag presentation and T cell and Ab-mediated immunity.

Ubiquitination of MHC Class II Is Required for Development of Regulatory but Not Conventional CD4+ T Cells.

MHC class II (MHC II) displays peptides at the cell surface, a process critical for CD4+ T cell development and priming. Ubiquitination is a mechanism that dictates surface MHC II with the attachment of a polyubiquitin chain to peptide-loaded MHC II, promoting its traffic away from the plasma membrane. In this study, we have examined how MHC II ubiquitination impacts the composition and function of both conventional CD4+ T cell and regulatory T cell (Treg) compartments. Responses were examined in two models of altered MHC II ubiquitination: MHCIIKRKI /KI mice that express a mutant MHC II unable to be ubiquitinated or mice that lack membrane-associated RING-CH 8 (MARCH8), the E3 ubiquitin ligase responsible for MHC II ubiquitination specifically in thymic epithelial cells. Conventional CD4+ T cell populations in thymus, blood, and spleen of MHCIIKRKI/KI and March8 -/- mice were largely unaltered. In MLRs, March8 -/-, but not MHCIIKRKI/KI, CD4+ T cells had reduced reactivity to both self- and allogeneic MHC II. Thymic Treg were significantly reduced in MHCIIKRKI/KI mice, but not March8 -/- mice, whereas splenic Treg were unaffected. Neither scenario provoked autoimmunity, with no evidence of immunohistopathology and normal levels of autoantibody. In summary, MHC II ubiquitination in specific APC types does not have a major impact on the conventional CD4+ T cell compartment but is important for Treg development.

Impact of the utilization of 500 mL IV bags on crystalloid resuscitation volumes administered to trauma patients.

INTRODUCTION: Administering large volumes of crystalloids to trauma patients has been shown to exacerbate metabolic complications of hemorrhage including dilutional coagulopathy and worsening acidosis The aim of this study was to evaluate crystalloid administration volumes in trauma patients after replacing 1 L IV containers with 500 mL IV containers in the emergency department trauma resuscitation bay. MATERIALS AND METHODS: This was a single-center, IRB-approved, retrospective cohort evaluation of adult trauma patients conducted at an 864-bed community tertiary referral center located in the southeastern United States. Patterns of crystalloid administration were examined before and after the trauma resuscitation bay began to exclusively stock 500 mL IV containers. The primary outcome was mean total crystalloid volume infused from time of injury to hospital admission. Secondary outcomes included mean total crystalloid volume infused prior to administration of blood products, proportion of patients who received less than 2 L total of crystalloids, time to initiation of blood products, and mortality in both the emergency department and in-hospital. RESULTS: Patient characteristics were largely similar between both groups including age, mechanism of injury, and Injury Severity Score. For the primary outcome, the mean total crystalloid volume infused from time of injury to hospital administration, patients in the 500 mL IV fluid container group were administered 555 mL less crystalloid when compared to the 1 L IV fluid container group, 1048 mL vs 1603 mL (p < 0.01; 95% CI 406 mL - 704 mL), respectively. After conversion to the 500 mL IV container bags, there was a 27.5% increase in the proportion of patients receiving less than 2 L of crystalloid, 90.5% vs 63.0% in the 500 mL IV fluid container and 1 L IV fluid container groups, respectively (p < 0.01). CONCLUSIONS: Due to reduced mortality, expanding literature and guidelines clearly support minimizing IV crystalloid resuscitation. Institutions must now work to minimize use of IV crystalloids to hemorrhaging trauma patients and a simple solution of using smaller IV fluid bags was shown to improve adherence to this practice.

Comparative hemostatic efficacy of 4F-PCC in patients with intracranial hemorrhage on factor Xa inhibitors versus warfarin.

OBJECTIVE: Patients experiencing an intracranial hemorrhage (ICH) on oral anticoagulants often require rapid reversal. This study evaluated patients taking factor Xa inhibitors or warfarin that received reversal with 4-factor prothrombin complex concentrate (4F-PCC) for an ICH. The objective of the study was to determine if the efficacy of 4F-PCC for the reversal of factor Xa inhibitors is noninferior to its use in warfarin reversal in patients with ICH. METHODS: This was a retrospective, single center, noninferiority trial. Patients presenting to the emergency department with ICH were divided into two cohorts: those taking factor Xa inhibitors versus those taking warfarin. In each cohort, patients received anticoagulation reversal with weight-based 4F-PCC. The primary endpoint was hemostatic efficacy defined as ≤20% expansion in hematoma volume on repeat computed tomography imaging. A pre-specified noninferiority margin of -10% was selected to evaluate the difference between groups for the primary endpoint. RESULTS: A total of 221 patients were included in the study (factor Xa inhibitors, n = 87; warfarin, n = 134). Effective hemostasis was achieved in 70 patients (81%) on factor Xa inhibitors compared to 111 patients (83%) on warfarin, (-2.4% difference, [95% confidence interval, -12.87 to 8.12]; p = 0.654). There was no statistically significant difference between groups with regards to the primary outcome; however, the use of 4F-PCC in factor Xa inhibitor reversal was not noninferior when compared to 4F-PCC use for warfarin reversal. Hospital length of stay and discharge disposition were similar between cohorts. CONCLUSIONS: The efficacy of 4F-PCC in reversing factor Xa inhibitor-related ICH compared to warfarin-related ICH was not significantly different between groups; however, these results did not prove noninferiority. Further study is warranted to delineate 4F-PCC's role in reversing factor Xa inhibitors in patients with ICH.