Emergency triage of brain computed tomography via anomaly detection with a deep generative model.

Triage is essential for the early diagnosis and reporting of neurologic emergencies. Herein, we report the development of an anomaly detection algorithm (ADA) with a deep generative model trained on brain computed tomography (CT) images of healthy individuals that reprioritizes radiology worklists and provides lesion attention maps for brain CT images with critical findings. In the internal and external validation datasets, the ADA achieved area under the curve values (95% confidence interval) of 0.85 (0.81-0.89) and 0.87 (0.85-0.89), respectively, for detecting emergency cases. In a clinical simulation test of an emergency cohort, the median wait time was significantly shorter post-ADA triage than pre-ADA triage by 294 s (422.5 s [interquartile range, IQR 299] to 70.5 s [IQR 168]), and the median radiology report turnaround time was significantly faster post-ADA triage than pre-ADA triage by 297.5 s (445.0 s [IQR 298] to 88.5 s [IQR 179]) (all p < 0.001).

Alginate-reinforced poly(3-hydroxybutyrate)/ poly(hydroxybutyrate-co-hydroxyvalerate) aerogel monoliths fabricated by phase separation as environmental floating adsorbents.

Poly(3-hydroxybutyrate) (PHB)/poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) aerogel monoliths were prepared via nonsolvent induced phase separation and then sequentially immersed in ethanol and sodium alginate (ALG) solutions. The resulting composite aerogels contained up to a 52 wt% fraction ALG, causing a remarkable increase in their compressive modulus and collapse strength from 0.3 MPa and 33 kPa to 4 MPa and 406 kPa, respectively, i.e., by 13/12 times. An increase in the ALG contents in the composite aerogels allowed them to effectively adsorb both water and soybean oil, according to pseudo-second-order adsorption kinetics. The highly porous composite aerogel acted as an efficient floating adsorbent for a cationic dye (i.e., methylene blue (MB)) in water. MB adsorption was found to be strongly dependent on ALG contents in the adsorbent, as well as operating parameters such as the initial concentration, pH, and temperature of MB solutions. MB adsorption is best described by the Langmuir isotherm and follows pseudo-second-order kinetics. Ca2+-crosslinking of ALG further increased compressive strength but significantly decreased MB adsorption capability following pseudo-first-order kinetics, implying a slow internal diffusion step for MB adsorption due to its tightened network structure relative to noncrosslinked adsorbents.

Chitosan-reinforced PHB hydrogel and aerogel monoliths fabricated by phase separation with the solvent-exchange method.

The poly(3-hydroxybutyrate) (PHB) organogel monoliths were prepared by nonsolvent-induced phase separation (NIPS). The NIPS-derived organogels were solvent-exchanged with chitosan (CS) solution, resulting in the successful loading of CS into PHB hydrogel. With increasing the CS content, the as-prepared composite hydrogels became syringe injectable with excellent thixotropy due to the increase in the gel network's hydrophilicity. The hydrogels were successfully freeze-dried into PHB/CS composite aerogels, which exhibited remarkably improved compressive modulus/collapse strength of 1.6 MPa/159 kPa compared to 0.5 MPa/31 kPa for pure PHB aerogels. The effect of CS on gel crystallization and structure was also investigated. The amphiphilic PHB/CS hydrogels effectively entrapped both hydrophilic/cationic doxorubicin (DOX) and hydrophobic/anionic indomethacin (IDM). The drug release behavior depended on the charge interactions between drugs and CS. The accelerated DOX release in acidic condition from injected hydrogels owing to the charge repulsion shows potential for a controlled and localized cancer therapy.

OASL1-Mediated Inhibition of Type I IFN Reduces Influenza A Infection-Induced Airway Inflammation by Regulating ILC2s.

PURPOSE: Three observations drove this study. First, 2'-5'-oligoadenylate synthetase-like protein (OASL) is a negative regulator of type I interferon (IFN). Second, type I IFN plays a central role during virus infections and the pathogenesis of various diseases, including asthma. Third, influenza A virus (IAV) causes non-eosinophilic asthma. To evaluate the potential relationships between OASL, type I IFN, and pulmonary innate immune cells in IAV-induced acute airway inflammation by using Oasl1-/- mice. METHODS: Asthma was induced in wild-type (WT) and Oasl1-/- mice with IAV or ovalbumin (OVA). Airway hyperreactivity (AHR) and immune cell infiltration in the bronchoalveolar lavage (BAL) fluids were measured. The immune cells in the lungs were analyzed by flow cytometry. To investigate the ability of type I IFN to shape the response of lung type 2 innate lymphoid cells (ILC2s), IFN-α was treated intratracheally. Plasmacytoid dendritic cells (pDCs) sorted from bone marrow and ILC2s sorted from lungs of naive mice were co-cultured with/without interferon-alpha receptor subunit 1 (IFNAR-1)-blocking antibodies. RESULTS: In the IAV-induced asthma model, Oasl1-/- mice developed greater AHR and immune cell infiltration in the BAL fluids than WT mice. This was not observed in OVA-induced asthma, a standard model of allergen-induced asthma. The lungs of infected Oasl1-/- mice also had elevated DC numbers and Ifna expression and depressed IAV-induced ILC2 responses, namely, proliferation and type 2 cytokine and amphiregulin production. Intratracheal administration of type I IFN in naïve mice suppressed lung ILC2 production of type 2 cytokines and amphiregulin. Co-culture of ILC2s with pDCs showed that pDCs inhibit the function of ILC2s by secreting type I IFN. CONCLUSIONS: OASL1 may impede the IAV-induced acute airway inflammation that drives AHR by inhibiting IAV-induced type I IFN production from lung DCs, thereby preserving the functions of lung ILC2s, including their amphiregulin production.

Double-Network Hydrogel Films Based on Cellulose Derivatives and κ-Carrageenan with Enhanced Mechanical Strength and Superabsorbent Properties.

Covalently crosslinked sodium carboxymethyl cellulose (CMC)-hydroxyethyl cellulose (HEC) hydrogel films were prepared using citric acid (CA) as the crosslinking agent. Thereafter, the physically crosslinked κ-carrageenan (κ-CG) polymer was introduced into the CMC-HEC hydrogel structure, yielding κ-CG/CMC-HEC double network (DN) hydrogels. The κ-CG physical network provided sacrificial bonding, which effectively dissipated the stretching energy, resulting in an increase in the tensile modulus, tensile strength, and fracture energy of the DN hydrogels by 459%, 305%, and 398%, respectively, compared with those of the CMC-HEC single network (SN) hydrogel. The dried hydrogels exhibited excellent water absorbency with a maximum water-absorption capacity of 66 g/g in distilled water. Compared with the dried covalent SN gel, the dried DN hydrogels exhibited enhanced absorbency under load, attributed to their improved mechanical properties. The water-absorption capacities and kinetics were dependent on the size of the dried gel and the pH of the water.

Chitosan-dipeptide hydrogels as potential anticancer drug delivery systems.

A novel chitosan-dipeptide hydrogel was fabricated through a combination of self-assembly of 9-fluorenylmethoxycarbonyl-modified diphenylalanine (Fmoc-FF) and its electrostatic interaction with glycol chitosan (GCS). Hydrogel strength and stability depended on its composition. The highest gel strength was observed at a Fmoc-FF mass fraction (ϕFF) of 0.85, whereby the highest combined strength of the two interactions was achieved. As the ϕFF increased above 0.6, gel stability decreased in buffered solution at pH 7.46. The incorporation of doxorubicin (DOX) as a cationic model drug significantly increased the stability of the complex hydrogels. DOX-loaded hydrogels exhibited slow DOX release, probably due to the drug's strong binding to Fmoc-FF via electrostatic attraction and the high gel stability. These hydrogels also exhibited excellent thixotropic features that facilitated the development of injectable self-healing drug delivery systems. Notably, DOX release was significantly accelerated as the pH of the medium decreased from 7.46 to 5.5 and 4.0, possibly due to hydrogel components' protonation. The DOX-loaded hydrogel exhibited notable cytotoxicity against A549 human lung cancer cells, which suggests the newly developed hydrogel to be a promising candidate vehicle for the localized and controlled drug delivery in cancer therapy.

Nonsolvent-induced phase separation of poly(3-hydroxybutyrate) and poly(hydroxybutyrate-co-hydroxyvalerate) blend as a facile platform to fabricate versatile nanofiber gels: Aero-, hydro-, and oleogels.

We demonstrated a strategy to prepare different types of 3-D nanofibrous polymeric gels, including hydro-, aero-, and oleogels by nonsolvent-induced phase separation (NIPS). NIPS-derived gel monoliths of poly(3-hydroxybutyrate) (PHB) and poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) blends were converted into hydrogels and aerogels by solvent exchange and freeze-drying, respectively. The high hydrophobicity and porosity of the nanofibrous PHB/PHBV aerogels enabled them to absorb various oils and swell to 20-30 times their own weight. The pseudo-second-order model was successfully used to describe the oil absorption behavior, and the obtained absorption rate constant increased with increasing PHBV content. The oil-swollen aerogels were highly elastic, thereby indicating that NIPS-derived aerogels are an excellent template for the fabrication of oleogels. With an increase in the PHBV ratio, the gels exhibited reduced modulus and collapse strength but increased collapse strain, thereby revealing higher ductility by compression. The rapid separation and re-binding of the liquid phase entrapped in the nanofiber network resulted in the unique thixotropic properties of the hydro- and oleogels. Indomethacin, a hydrophobic model drug, was successfully incorporated into injectable self-healing oleogels containing soybean oil and aerogels. These gels exhibited excellent cytocompatibility, and a better sustained drug release was observed for the oleogels compared to the aerogels.

Nanofibrous Foams of Poly(3-hydroxybutyrate)/Cellulose Nanocrystal Composite Fabricated Using Nonsolvent-Induced Phase Separation.

In this study, we fabricated nanofibrous foams of neat poly(3-hydroxybutyrate) (PHB) and PHB/cellulose nanocrystal (CNC) nanocomposite using nonsolvent-induced phase separation (NIPS) followed by solvent extraction. Two different nonsolvents, tetrahydrofuran (THF) and 1,4-dioxane (Diox), in combination with the solvent, chloroform (CF), were used for NIPS. The parameters of NIPS-derived crystallization kinetics were calculated using Avrami analysis of time-dependent infrared spectral measurements. The lower viscosity and poorer PHB affinity of THF than those of Diox resulted in rapid crystallization and gelation rate, which in turn resulted in higher strength of the foam. The mechanical reinforcement by the incorporation of CNCs was achieved for the composite foam prepared in Diox/CF but not in THF/CF, owing to the relatively better dispersion of the CNCs in Diox than that in THF. A rapid rate of NIPS-derived crystallization and gelation was achieved in THF/CF with the incorporation of CNCs, indicating the effective crystal nucleation of CNCs. However, the presence of CNCs deaccelerated the crystallization in Diox/CF, indicating that the inhibition effect of PHB mobility became more dominant than the nucleation effect of CNCs; this was because the CNC dispersion became more homogeneous in Diox/CF. In vitro cell viability assays exhibited excellent cytocompatibility of the foams, thereby showing potential for use in biomedical applications.

Porous Poly(3-hydroxybutyrate) Scaffolds Prepared by Non-Solvent-Induced Phase Separation for Tissue Engineering.

Highly porous poly(3-hydroxybutyrate) (PHB) scaffolds were fabricated using non-solvent-induced phase separation with chloroform as the solvent and tetrahydrofuran as the non-solvent. The microporosity, nanofiber morphology, and mechanical strength of the scaffolds were adjusted by varying the fabrication parameters, such as the polymer concentration and solvent composition. The influence of these parameters on the structure and morphology of PHB organogels and scaffolds was elucidated using small-angle neutron scattering and scanning electron microscopy. The organogels and scaffolds in this study have a complex hierarchical structure, extending over a wide range of length scales. In vitro viability assays were performed using the human keratinocyte cell line (HaCaT), and all PHB scaffolds demonstrated the excellent cell viability. Microporosity had the greatest impact on HaCaT cell proliferation on PHB scaffolds, which was determined after a 3-day incubation period with scaffolds of different morphologies and mechanical properties. The superior cell viability and the controlled scaffold properties and morphologies suggested PHB scaffolds fabricated by non-solvent-induced phase separation using chloroform and tetrahydrofuran as promising biomaterials for the applications of tissue engineering, particularly of epidermal engineering. Electronic Supplementary Material: Supplementary material is available in the online version of this article at 10.1007/s13233-020-8109-x.

OASL1 Traps Viral RNAs in Stress Granules to Promote Antiviral Responses.

Oligoadenylate synthetase (OAS) protein family is the major interferon (IFN)-stimulated genes responsible for the activation of RNase L pathway upon viral infection. OAS-like (OASL) is also required for inhibition of viral growth in human cells, but the loss of one of its mouse homolog, OASL1, causes a severe defect in termination of type I interferon production. To further investigate the antiviral activity of OASL1, we examined its subcellular localization and regulatory roles in IFN production in the early and late stages of viral infection. We found OASL1, but not OASL2, formed stress granules trapping viral RNAs and promoted efficient RLR signaling in early stages of infection. Stress granule formation was dependent on RNA binding activity of OASL1. But in the late stages of infection, OASL1 interacted with IRF7 transcripts to inhibit translation resulting in down regulation of IFN production. These results implicate that OASL1 plays context dependent functions in the antiviral response for the clearance and resolution of viral infections.

Integrin CD11b negatively regulates Mincle-induced signaling via the Lyn-SIRPα-SHP1 complex.

During mycobacteria infection, anti-inflammatory responses allow the host to avoid tissue damage caused by overactivation of the immune system; however, little is known about the negative modulators that specifically control mycobacteria-induced immune responses. Here we demonstrate that integrin CD11b is a critical negative regulator of mycobacteria cord factor-induced macrophage-inducible C-type lectin (Mincle) signaling. CD11b deficiency resulted in hyperinflammation following mycobacterial infection. Activation of Mincle by mycobacterial components turns on not only the Syk signaling pathway but also CD11b signaling and induces formation of a Mincle-CD11b signaling complex. The activated CD11b recruits Lyn, SIRPα and SHP1, which dephosphorylate Syk to inhibit Mincle-mediated inflammation. Furthermore, the Lyn activator MLR1023 effectively suppressed Mincle signaling, indicating the possibility of Lyn-mediated control of inflammatory responses. These results describe a new role for CD11b in fine-tuning the immune response against mycobacterium infection.

Mycobacterial cord factor enhances migration of neutrophil-like HL-60 cells by prolonging AKT phosphorylation.

Trehalose 6,6'-dimycolate (TDM), or cord factor, is a crucial stimulus of immune responses during Mycobacterium tuberculosis infection. Although TDM has immuno-stimulatory properties, including adjuvant activity and the ability to induce granuloma formation, the mechanisms underlying these remain unknown. We hypothesized that TDM stimulates transendothelial migration of neutrophils, which are the first immune cells to infiltrate the tissue upon infection. In this study, it was shown that TDM enhances N-formylmethionyl-leucyl-phenylalanine (fMLP)-induced chemotaxis and transendothelial movement by prolonging AKT phosphorylation in human neutrophils. TDM induced expression of macrophage-inducible C-type lectin, a receptor for TDM, and induced secretion of pro-inflammatory cytokines and chemokines in differentiated HL-60 cells. In 2- and 3-D neutrophil migration assays, TDM-stimulated neutrophils showed increased fMLP-induced chemotaxis and transendothelial migration. Interestingly, following fMLP stimulation of TDM-activated neutrophils, AKT, a crucial kinase for neutrophil polarization and chemotaxis, showed prolonged phosphorylation at serine 473. Taken together, these data suggest that TDM modulates transendothelial migration of neutrophils upon mycobacterial infection through prolonged AKT phosphorylation. AKT may therefore be a promising therapeutic target for enhancing immune responses to mycobacterial infection.

Macrophage C-type lectin is essential for phagosome maturation and acidification during Escherichia coli-induced peritonitis.

Sepsis is a life-threatening condition caused by an uncontrolled response to bacterial infection. Impaired bactericidal activity in the host is directly associated with severe sepsis; however, the underlying regulatory mechanism(s) is largely unknown. Here, we show that MCL (macrophage C-type lectin) plays a crucial role in killing bacteria during Escherichia coli-induced peritonitis. MCL-deficient mice with E. coli-induced sepsis showed lower survival rates and reduced bacterial clearance when compared with control mice, despite similar levels of proinflammatory cytokine production. Although the ability of macrophages from MCL-deficient mice to kill bacteria was impaired, they showed normal phagocytic activity and production of reactive oxygen species. In addition, MCL-deficient macrophages showed defective phagosome maturation and phagosomal acidification after E. coli infection. Taken together, these results indicate that MCL plays an important role in host defense against E. coli infection by promoting phagosome maturation and acidification, thereby providing new insight into the role of MCL during pathogenesis of sepsis and offering new therapeutic options.

Mincle activation enhances neutrophil migration and resistance to polymicrobial septic peritonitis.

Sepsis is a systemic inflammatory response to bacterial infection. The therapeutic options for treating sepsis are limited. Impaired neutrophil recruitment into the infection site is directly associated with severe sepsis, but the precise mechanism is unclear. Here, we show that Mincle plays a key role in neutrophil migration and resistance during polymicrobial sepsis. Mincle-deficient mice exhibited lower survival rates in experimental sepsis from cecal ligation and puncture and Escherichia coli-induced peritonitis. Mincle deficiency led to higher serum inflammatory cytokine levels and reduced bacterial clearance and neutrophil recruitment. Transcriptome analyses revealed that trehalose dimycolate, a Mincle ligand, reduced the expression of G protein-coupled receptor kinase 2 (GRK2) in neutrophils. Indeed, GRK2 expression was upregulated, but surface expression of the chemokine receptor CXCR2 was downregulated in blood neutrophils from Mincle-deficient mice with septic injury. Moreover, CXCL2-mediated adhesion, chemotactic responses, and F-actin polymerization were reduced in Mincle-deficient neutrophils. Finally, we found that fewer Mincle-deficient neutrophils infiltrated from the blood circulation into the peritoneal fluid in bacterial septic peritonitis compared with wild-type cells. Thus, our results indicate that Mincle plays an important role in neutrophil infiltration and suggest that Mincle signaling may provide a therapeutic target for treating sepsis.

Mincle-mediated translational regulation is required for strong nitric oxide production and inflammation resolution.

In response to persistent mycobacteria infection, the host induces a granuloma, which often fails to eradicate bacteria and results in tissue damage. Diverse host receptors are required to control the formation and resolution of granuloma, but little is known concerning their regulatory interactions. Here we show that Mincle, the inducible receptor for mycobacterial cord factor, is the key switch for the transition of macrophages from cytokine expression to high nitric oxide production. In addition to its stimulatory role on TLR-mediated transcription, Mincle enhanced the translation of key genes required for nitric oxide synthesis through p38 and eIF5A hypusination, leading to granuloma resolution. Thus, Mincle has dual functions in the promotion and subsequent resolution of inflammation during anti-mycobacterial defence using both transcriptional and translational controls.

Oligoadenylate synthase-like (OASL) proteins: dual functions and associations with diseases.

The study of antiviral pathways to reveal methods for the effective response and clearance of virus is closely related to understanding interferon (IFN) signaling and its downstream target genes, IFN-stimulated genes. One of the key antiviral factors induced by IFNs, 2'-5' oligoadenylate synthase (OAS), is a well-known molecule that regulates the early phase of viral infection by degrading viral RNA in combination with RNase L, resulting in the inhibition of viral replication. In this review, we describe OAS family proteins from a different point of view from that of previous reviews. We discuss not only RNase L-dependent (canonical) and -independent (noncanonical) pathways but also the possibility of the OAS family members as biomarkers for various diseases and clues to non-immunological functions based on recent studies. In particular, we focus on OASL, a member of the OAS family that is relatively less well understood than the other members. We will explain its anti- and pro-viral dual roles as well as the diseases related to single-nucleotide polymorphisms in the corresponding gene.

Genome-wide profiling of in vivo LPS-responsive genes in splenic myeloid cells.

Lipopolysaccharide (LPS), the major causative agent of bacterial sepsis, has been used by many laboratories in genome-wide expression profiling of the LPS response. However, these studies have predominantly used in vitro cultured macrophages (Macs), which may not accurately reflect the LPS response of these innate immune cells in vivo. To overcome this limitation and to identify inflammatory genes in vivo, we have profiled genome-wide expression patterns in non-lymphoid, splenic myeloid cells extracted directly from LPS-treated mice. Genes encoding factors known to be involved in mediating or regulating inflammatory processes, such as cytokines and chemokines, as well as many genes whose immunological functions are not well known, were strongly induced by LPS after 3 h or 8 h of treatment. Most of the highly LPS-responsive genes that we randomly selected from the microarray data were independently confirmed by quantitative RT-PCR, implying that our microarray data are quite reliable. When our in vivo data were compared to previously reported microarray data for in vitro LPS-treated Macs, a significant proportion (∼20%) of the in vivo LPS-responsive genes defined in this study were specific to cells exposed to LPS in vivo, but a larger proportion of them (∼60%) were influenced by LPS in both in vitro and in vivo settings. This result indicates that our in vivo LPS-responsive gene set includes not only previously identified in vitro LPS-responsive genes but also novel LPS-responsive genes. Both types of genes would be a valuable resource in the future for understanding inflammatory responses in vivo.

Neutrophils Promote Mycobacterial Trehalose Dimycolate-Induced Lung Inflammation via the Mincle Pathway.

Trehalose 6,6'-dimycolate (TDM), a cord factor of Mycobacterium tuberculosis (Mtb), is an important regulator of immune responses during Mtb infections. Macrophages recognize TDM through the Mincle receptor and initiate TDM-induced inflammatory responses, leading to lung granuloma formation. Although various immune cells are recruited to lung granulomas, the roles of other immune cells, especially during the initial process of TDM-induced inflammation, are not clear. In this study, Mincle signaling on neutrophils played an important role in TDM-induced lung inflammation by promoting adhesion and innate immune responses. Neutrophils were recruited during the early stage of lung inflammation following TDM-induced granuloma formation. Mincle expression on neutrophils was required for infiltration of TDM-challenged sites in a granuloma model induced by TDM-coated-beads. TDM-induced Mincle signaling on neutrophils increased cell adherence by enhancing F-actin polymerization and CD11b/CD18 surface expression. The TDM-induced effects were dependent on Src, Syk, and MAPK/ERK kinases (MEK). Moreover, coactivation of the Mincle and TLR2 pathways by TDM and Pam3CSK4 treatment synergistically induced CD11b/CD18 surface expression, reactive oxygen species, and TNFα production by neutrophils. These synergistically-enhanced immune responses correlated with the degree of Mincle expression on neutrophil surfaces. The physiological relevance of the Mincle-mediated anti-TDM immune response was confirmed by defective immune responses in Mincle⁻/⁻ mice upon aerosol infections with Mtb. Mincle-mutant mice had higher inflammation levels and mycobacterial loads than WT mice. Neutrophil depletion with anti-Ly6G antibody caused a reduction in IL-6 and monocyte chemotactic protein-1 expression upon TDM treatment, and reduced levels of immune cell recruitment during the initial stage of infection. These findings suggest a new role of Mincle signaling on neutrophils during anti-mycobacterial responses.