Dectin-1 Controls TLR9 Trafficking to Phagosomes Containing ß-1,3 Glucan.

Dectin-1 and TLR9 play distinct roles in the recognition and induction of innate immune responses to Aspergillus fumigatus and Candida albicans. Dectin-1 is a receptor for the major fungal cell wall carbohydrate ß-1,3 glucan that induces inflammatory cytokines and controls phagosomal maturation through spleen tyrosine kinase activation. TLR9 is an endosomal TLR that also modulates the inflammatory cytokine response to fungal pathogens. In this study, we demonstrate that ß-1,3 glucan beads are sufficient to induce dynamic redistribution and accumulation of cleaved TLR9 to phagosomes. Trafficking of TLR9 to A. fumigatus and C. albicans phagosomes requires Dectin-1 recognition. Inhibition of phagosomal acidification blocks TLR9 accumulation on phagosomes containing ß-1,3 glucan beads. Dectin-1-mediated spleen tyrosine kinase activation is required for TLR9 trafficking to ß-1,3 glucan-, A. fumigatus-, and C. albicans-containing phagosomes. In addition, Dectin-1 regulates TLR9-dependent gene expression. Collectively, our study demonstrates that recognition of ß-1,3 glucan by Dectin-1 triggers TLR9 trafficking to ß-1,3 glucan-containing phagosomes, which may be critical in coordinating innate antifungal defense.

Dectin-1-dependent LC3 recruitment to phagosomes enhances fungicidal activity in macrophages.

Autophagy has been postulated to play role in mammalian host defense against fungal pathogens, although the molecular details remain unclear. Here, we show that primary macrophages deficient in the autophagic factor LC3 demonstrate diminished fungicidal activity but increased cytokine production in response to Candida albicans stimulation. LC3 recruitment to fungal phagosomes requires activation of the fungal pattern receptor dectin-1. LC3 recruitment to the phagosome also requires Syk signaling but is independent of all activity by Toll-like receptors and does not require the presence of the adaptor protein Card9. We further demonstrate that reactive oxygen species generation by NADPH oxidase is required for LC3 recruitment to the fungal phagosome. These observations directly link LC3 to the inflammatory pathway against C. albicans in macrophages.

Dectin-1 activation controls maturation of ß-1,3-glucan-containing phagosomes.

Elimination of fungal pathogens by phagocytes requires phagosome maturation, a process that involves the recruitment and fusion of intracellular proteins. The role of Dectin-1, a ß-1,3-glucan receptor, critical for fungal recognition and triggering of Th17 responses, to phagosomal maturation has not been defined. We show that GFP-Dectin-1 translocates to the fungal phagosome, but its signal decays after 2 h. Inhibition of acidification results in retention of GFP-Dectin-1 to phagosome membranes highlighting the requirement for an acidic pH. Following ß-1,3-glucan recognition, GFP-Dectin-1 undergoes tyrosine phosphorylation by Src kinases with subsequent Syk activation. Our results demonstrate that Syk is activated independently of intraphagosomal pH. Inhibition of Src or Syk results in prolonged retention of GFP-Dectin-1 to the phagosome signifying a link between Syk and intraphagosomal pH. ß-1,3-glucan phagosomes expressing a signaling incompetent Dectin-1 failed to mature as demonstrated by prolonged Dectin-1 retention, presence of Rab5B, failure to acquire LAMP-1 and inability to acidify. Phagosomes containing Candida albicans also require Dectin-1-dependent Syk activation for phagosomal maturation. Taken together, these results support a model where Dectin-1 not only controls internalization of ß-1,3-glucan containing cargo and triggers proinflammatory cytokines, but also acts as a master regulator for subsequent phagolysosomal maturation through Syk activation.

Effects of side group functionality and molecular weight on the activity of synthetic antimicrobial polypeptides.

The rapid emergence of antibiotic-resistant bacteria along with increasing difficulty in biofilm treatment has caused an immediate need for the development of new classes of antimicrobial therapeutics. We have developed a library of antimicrobial polypeptides, prepared by the ring-opening polymerization of γ-propargyl-L-glutamate N-carboxyanhydride and the alkyne-azide cycloaddition click reaction, which mimic the favorable characteristics of naturally occurring antimicrobial peptides (AmPs). AmPs are known not to cause drug resistance as well as prevent bacteria attachment on surfaces. The ease and scale of synthesis of the antimicrobial polypeptides developed here are significantly improved over the traditional Merrifield synthetic peptide approaches needed for naturally occurring antimicrobial peptides and avoids the unique challenges of biosynthetic pathways. The polypeptides range in length from 30 to 140 repeat units and can have varied side group functionality, including primary, secondary, tertiary, and quaternary amines with hydrocarbon side chains ranging from 1 to 12 carbons long. Overall, we find these polypeptides to exhibit broad-spectrum activity against both Gram positive and Gram negative bacteria, namely, S. aureus and E. coli , while having very low hemolytic activity. Many of the polypeptides can also be used as surface coatings to prevent bacterial attachment. The polypeptide library developed in this work addresses the need for effective biocompatible therapeutics for drug delivery and medical device coatings.

Comparison of Resident and Faculty Screening for Social Determinants of Health in an Academic Pediatric Primary Care Practice.

BACKGROUND: Social determinants of health (SDH) have an important role in children's health and development and should be investigated in pediatric well child care. METHODS: A retrospective chart review of children aged 5-17 at well visits at an urban academic pediatric primary care practice was performed. Chi-square tests of independence and z-test for proportions were used to assess differences between residents and faculty SDH screening.  Results: Faculty screened for SDH more frequently than residents (P<0.05). Residents screened less frequently for food insecurity (P<0.05) and financial insecurity (P<0.05). Financial insecurity was endorsed less frequently by resident families (P<0.05), while school absence was endorsed more frequently by resident families (P<0.05). Referrals to the clinic's community resource desk did not differ between residents and faculty. CONCLUSIONS: Differences exist in screening and need between clinician groups. Despite these differences, there was no difference in community resource desk referrals.

A Novel, Trauma-Informed Physical Examination Curriculum for First-Year Medical Students.

Introduction: Trauma is prevalent in the general population in various forms and has lasting effects on health. Physicians routinely examine patients who have experienced trauma, although most providers lack training in trauma-informed care, a well-established framework for providing quality care to trauma survivors. To address this gap, we implemented a novel curriculum on trauma-informed physical examination skills for first-year medical students. Methods: We held a large-group lecture for 148 first-year medical students and 40 faculty members to introduce a framework for a trauma-informed physical examination, using a standardized patient for demonstration. The framework included specific language and behaviors to employ before, during, and after the examination in order to enhance patients' sense of safety, control, and trust. Students then transitioned to small groups to practice performing vital signs using a trauma-informed approach, with supervision from MD faculty. Results: Five-point scales were used to evaluate students' knowledge gained from the session and satisfaction with the session. Overall satisfaction with the session was rated as 4.08 (SD = 0.81), and students felt that the session was highly effective in defining a trauma-informed physical examination (4.29, SD = 0.70). Discussion: The session was well received and effective in teaching future physicians trauma-informed skills. We offer other institutions a model for incorporating trauma-informed care into clinical skills curricula.

Vancomycin storage stability in multilayer thin film coatings for on-demand care.

A rise in drug-resistant bacteria coupled with a decline in the development of new classes of antibiotics has increased the need for new local antibiotic delivery methods. To address this need, we have previously developed layer-by-layer assembled films releasing the potent antibiotic, vancomycin, for use as coatings for on-demand care. In this work, we explore the potential for practical application of these films through an in-depth study of their long term storage stability over a wide range of temperatures, including refrigerator, room and elevated temperature (4, 25 and 37°C, respectively). Our results indicate that the drug-release profiles and minimum inhibitory concentration against Staphylococcus aureus of vancomycin released from films stored at these different conditions are not affected over a period of 1-6 months. Consequently, these films can be used where traditional refrigeration of prepared intravenous vancomycin solutions or immediate reconstitution of lyophilized vancomycin is not possible.

Release of vancomycin from multilayer coated absorbent gelatin sponges.

Wounds have the potential to become infected during any surgical procedure. Gelatin sponges that are commonly used to absorb blood during invasive surgeries would benefit tremendously if they released antibiotics. In this work, we have examined coating a commercial gelatin sponge with degradable polymer multilayer films containing vancomycin. The effect of the film on sponge absorption capabilities and the effect of the sponge on drug release kinetics were both examined. Application of vancomycin containing layer-by-layer assembled films to this highly porous substrate greatly increased drug loading up to approximately 880% compared to a flat substrate. Vancomycin drug release was extended out to 6 days compared to 2 days for film coated flat substrates. Additionally, the absorbent properties of the gelatin sponge were actually enhanced by up to 170% due to the presence of the vancomycin film coating. A comparison of film coated sponges with sponges soaked directly in vancomycin demonstrated the ability of the multilayer films to control drug release. Film released vancomycin was also found to remain highly therapeutic with unchanged antimicrobial properties compared to the neat drug, demonstrated by quantifying vancomycin activity against Staphylococcus aureus in vitro.

Hemostatic multilayer coatings.

Spray layer-by-layer assembly is used to create hemostatic films containing thrombin and tannic acid. The spray assembly technique enables coating of porous and absorbent commercial gelatin sponges with these films. Coated sponges are able to promote instantaneous hemostasis in a porcine spleen bleeding model.