An Allosteric Anti-tryptase Antibody for the Treatment of Mast Cell-Mediated Severe Asthma.

Severe asthma patients with low type 2 inflammation derive less clinical benefit from therapies targeting type 2 cytokines and represent an unmet need. We show that mast cell tryptase is elevated in severe asthma patients independent of type 2 biomarker status. Active ß-tryptase allele count correlates with blood tryptase levels, and asthma patients carrying more active alleles benefit less from anti-IgE treatment. We generated a noncompetitive inhibitory antibody against human ß-tryptase, which dissociates active tetramers into inactive monomers. A 2.15 Å crystal structure of a ß-tryptase/antibody complex coupled with biochemical studies reveal the molecular basis for allosteric destabilization of small and large interfaces required for tetramerization. This anti-tryptase antibody potently blocks tryptase enzymatic activity in a humanized mouse model, reducing IgE-mediated systemic anaphylaxis, and inhibits airway tryptase in Ascaris-sensitized cynomolgus monkeys with favorable pharmacokinetics. These data provide a foundation for developing anti-tryptase as a clinical therapy for severe asthma.

Perceived Benefits of Peer Support Groups for Stroke Survivors and Caregivers in Rural North Carolina.

BACKGROUND Significant geographical disparities exist in stroke prevalence among southeastern states, including North Carolina. Additionally, stroke is more prevalent in rural areas. Peer support groups play an important role in stroke recovery by providing tools for effective coping, alleviating psychological stress, and creating an outlet for stroke survivors and caregivers. However, their perceived benefits have not been clearly defined for rural stroke survivors and their families.METHODS This qualitative study describes the experiences of survivors and caregivers in rural North Carolina who have participated in stroke peer support groups. Four focus groups were conducted with 32 participants (average age 67 years, 72% female) in 4 rural North Carolina counties, using a semi-structured discussion guide and an inductive coding approach.RESULTS Thematic analysis revealed that participants in rural support groups seek and receive knowledge from their support groups and feel empowered by providing and receiving this knowledge. Shared experiences cultivate a sense of community, and participants viewed support outside of the support group as necessary to their recovery process.LIMITATIONS This study reflects the views of a small group of predominantly non-Hispanic, white stroke survivors and caregivers who voluntarily participated. We did not conduct separate focus groups with survivors and caregivers.CONCLUSION Peer support groups are a sparse, but critical resource for rural stroke survivors and caregivers because they provide information and community that can assist with recovery to health and independence.

Cytochrome P450-derived epoxyeicosatrienoic acids and coronary artery disease in humans: a targeted metabolomics study.

Cytochrome P450 (CYP)-derived epoxyeicosatrienoic acids (EETs) exhibit potent cardiovascular protective effects in preclinical models, and promoting the effects of EETs has emerged as a potential therapeutic strategy for coronary artery disease (CAD). The relationship between circulating EET levels and CAD extent in humans, however, remains unknown. A panel of free (unesterified) plasma eicosanoid metabolites was quantified in 162 patients referred for coronary angiography, and associations with extent of CAD [no apparent CAD (N = 39), nonobstructive CAD (N = 51), and obstructive CAD (N = 72)] were evaluated. A significant relationship between free EET levels and CAD extent was observed (P = 0.003) such that the presence of obstructive CAD was associated with lower circulating EET levels. This relationship was confirmed in multiple regression analysis where CAD extent was inversely and significantly associated with EET levels (P = 0.013), and with a biomarker of EET biosynthesis (P < 0.001), independent of clinical and demographic factors. Furthermore, quantitative enrichment analysis revealed that these associations were the most pronounced compared with other eicosanoid metabolism pathways. Collectively, these findings suggest that the presence of obstructive CAD is associated with lower EET metabolite levels secondary to suppressed EET biosynthesis. Novel strategies that promote the effects of EETs may have therapeutic promise for patients with obstructive CAD.

The PLUNGE randomized controlled trial: evaluation of a games-based physical activity professional learning program in primary school physical education.

OBJECTIVE: To evaluate the efficacy of the Professional Learning for Understanding Games Education (PLUNGE) program on fundamental movement skills (FMS), in-class physical activity and perceived sporting competence. METHODS: A cluster-randomized controlled trial involving one year six class each from seven primary schools (n=168; mean age=11.2 years, SD=1.0) in the Hunter Region, NSW, Australia. In September (2013) participants were randomized by school into the PLUNGE intervention (n=97 students) or the 7-week wait-list control (n=71) condition. PLUNGE involved the use of Game Centered curriculum delivered via an in-class teacher mentoring program. Students were assessed at baseline and 8-week follow-up for three object control FMS (Test of Gross Motor Development 2), in-class physical activity (pedometer steps/min) and perceived sporting competence (Self-perception Profile for Children). RESULTS: Linear mixed models revealed significant group-by-time intervention effects (all p<0.05) for object control competency (effect size: d=0.9), and in-class pedometer steps/min (d=1.0). No significant intervention effects (p>0.05) were observed for perceived sporting competence. CONCLUSIONS: The PLUNGE intervention simultaneously improved object control FMS proficiency and in-class PA in stage three students.

Predictors of Delayed Postoperative Respiratory Depression Assessed from Naloxone Administration.

BACKGROUND: The aim of this study was to identify patient and procedural characteristics associated with postoperative respiratory depression or sedation requiring naloxone intervention. METHODS: We identified patients who received naloxone to reverse opioid-induced respiratory depression or sedation within 48 hours after discharge from anesthetic care (transfer from the postanesthesia care unit or transfer from the operating room to postoperative areas) between July 1, 2008, and June 30, 2010. Patients were matched to 2 control subjects based on age, sex, and exact type of procedure performed during the same year. A chart review was performed to identify patient, anesthetic, and surgical factors that may be associated with risk for intervention requiring naloxone. In addition, we identified all patients who developed adverse respiratory events (hypoventilation, apnea, oxyhemoglobin desaturation, pain/sedation mismatch) during phase 1 anesthesia recovery. We performed conditional logistic regression taking into account the 1:2 matched set case-control study design to assess patient and procedural characteristics associated with naloxone use. RESULTS: We identified 134 naloxone administrations, 58% within 12 hours of discharge from anesthesia care, with an incidence of 1.6 per 1000 (95% confidence interval [CI], 1.3-1.9) anesthetics. The presence of obstructive sleep apnea (odds ratio [OR] = 2.45; 95% CI, 1.27-4.66; P = 0.008) and diagnosis of an adverse respiratory event in the postanesthesia recovery room (OR = 5.11; 95% CI, 2.32-11.27; P < 0.001) were associated with an increased risk for requiring naloxone to treat respiratory depression or sedation after discharge from anesthesia care. After discharge from anesthesia care, patients administered naloxone used a greater median dose of opioids (10 [interquartile range, 0-47.1] vs 5 [0-24.8] IV morphine equivalents, P = 0.020) and more medications with sedating side effects (n = 41 [31%] vs 24 [9%]; P < 0.001). CONCLUSIONS: Obstructive sleep apnea and adverse respiratory events in the recovery room are harbingers of increased risk for respiratory depression or sedation requiring naloxone after discharge from anesthesia care. Also, patients administered naloxone received more opioids and other sedating medications after discharge from anesthetic care. Our findings suggest that these patients may benefit from more careful monitoring after being discharged from anesthesia care.

LUZ-Y, a novel platform for the mammalian cell production of full-length IgG-bispecific antibodies.

The ability of bispecific antibodies to simultaneously bind two unique antigens has great clinical potential. However, most approaches utilized to generate bispecific antibodies yield antibody-like structures that diverge significantly from the structure of archetype human IgG, and those that do approach structural similarity to native antibodies are often challenging to engineer and manufacture. Here, we present a novel platform for the mammalian cell production of bispecific antibodies that differ from their parental mAbs by only a single point mutation per heavy chain. Central to this platform is the addition of a leucine zipper to the C terminus of the C(H)3 domain of the antibody that is sufficient to drive the heterodimeric assembly of antibody heavy chains and can be readily removed post-purification. Using this approach, we developed various antibody constructs including one-armed Abs, bispecific antibodies that utilize a common light chain, and bispecific antibodies that pair light chains to their cognate heavy chains via peptide tethers. We have applied this technology to various antibody pairings and will demonstrate the engineering, purification, and biological activity of these antibodies herein.

Amphiphilic dendrons as supramolecular holdase chaperones.

The aggregation of incompletely or incorrectly folded proteins is implicated in diseases including Alzheimer's, cataracts, and other maladies. Natural systems express protein chaperones to prevent or even reverse harmful protein aggregation. Synthetic chaperone-like systems have sought to mimic the action of their biological counterparts but typically require substantial optimization and high concentrations to be functional, or lack programmability that would enable the targeting of specific protein substrates. Here we report a series of amphiphilic dendrons that undergo assembly and inhibit the aggregation of fragment 16-22 amyloid ß protein (Aß16-22). We show that monodisperse dendrons with hydrophilic tetraethylene glycol chains and a hydrophobic core based on naphthyl and benzyl ethers undergo supramolecular assembly in aqueous solutions to form sphere-like particles. The solubility of these dendrons and their assemblies is tuned by varying the relative sizes of their hydrophilic and hydrophobic regions. Two water-soluble dendrons are discovered and shown, via fluorescence experiments with rhodamine 6G, to generate a hydrophobic environment. Furthermore, we demonstrate that sub-stoichiometric concentrations of these amphiphilic dendrons stabilize Aß16-22 peptide with respect to aggregation, mimicking the activity of holdase chaperones. Our results highlight the potential of these amphiphilic molecules as the basis for a novel approach to artificial chaperones that may address many of the challenges associated with existing synthetic chaperone mimics.

Debridement Versus Simple Scrubbing of External Fixator Pin Sites.

Introduction: Irrigation and debridement of external fixator pin sites are methods utilized by some orthopedic surgeons to minimize the risk of surgical site infections in patients undergoing definitive internal fixation after temporization in an external fixation device. This study aimed to determine if irrigation and debridement of external fixator pin sites leads to fewer deep surgical site infections, compared to simply scrubbing the external fixator pin sites with a chlorhexidine scrub-brush. Methods: This single center retrospective cohort study was performed at a university level I trauma center. All cases in which a single surgeon removed an external fixator and followed this with definitive open reduction and internal fixation (ORIF) in the same operative setting between October 2007 and October 2018 were reviewed. A total of 313 patients were temporized in 334 external fixators prior to ORIF and were included in the study. Results: Eighteen of the 179 Irrigation and Debridement cohort (10.0%) and 8 of the 155 Simple Scrubbing cohort (5.2%) had infections that required a return to the operating room. No statistical difference (p = 0.10) or meaningful effect size (Cohen's d = 0.18) were found between irrigation and debridement and simple scrubbing of external fixator pin sites. Conclusions: Given no significant differences were found in deep infection rates between debridement of pin sites versus simply scrubbing, it is reasonable to ask whether the time and resources required for debriding external fixator pin sites is worthwhile.

Localized Charge on Surfactant-Wrapped Single-Walled Carbon Nanotubes.

As-synthesized, semiconducting single-walled carbon nanotubes (SWCNTs) are nominally charge neutral. However, ionic surfactants that are commonly used to disperse SWCNTs in solution can lead to significantly charged aggregates adsorbed to the nanotube. Here, electrostatic force microscopy (EFM) was used to characterize the static-charge interactions between individual SWCNTs and the local environment. We report nonuniform spatial charge distributions with highly varying magnitudes ranging between ±15 e associated with surfactant coverage on long SWCNTs (>1.5 µm). EFM images acquired after resonant photoexcitation demonstrate charge carrier localization due to electrostatic interactions with charged surfactant aggregates. Charge densities as measured by EFM are used to estimate the depth of this electrostatically induced potential well, calculated to be on the order of hundreds of millielectronvolts, suggesting that surfactant charges heterogeneously covering SWCNTs provide traps for excitons potentially leading to their localization.

Tetravalent biepitopic targeting enables intrinsic antibody agonism of tumor necrosis factor receptor superfamily members.

Agonism of members of the tumor necrosis factor receptor superfamily (TNFRSF) with monoclonal antibodies is of high therapeutic interest due to their role in immune regulation and cell proliferation. A major hurdle for pharmacologic activation of this receptor class is the requirement for high-order clustering, a mechanism that imposes a reliance in vivo on Fc receptor-mediated crosslinking. This extrinsic dependence represents a potential limitation of virtually the entire pipeline of agonist TNFRSF antibody drugs, of which none have thus far been approved or reached late-stage clinical trials. We show that tetravalent biepitopic targeting enables robust intrinsic antibody agonism for two members of this family, OX40 and DR5, that is superior to extrinsically crosslinked native parental antibodies. Tetravalent biepitopic anti-OX40 engagement co-stimulated OX40low cells, obviated the requirement for CD28 co-signal for T cell activation, and enabled superior pharmacodynamic activity relative to native IgG in a murine vaccination model. This work establishes a proof of concept for an engineering approach that addresses a major gap for the therapeutic activation of this important receptor class.

Evaluation of a Longitudinal Institutional Advanced Pharmacy Practice Model.

OBJECTIVE: To evaluate a longitudinal experiential training model for advanced pharmacy practice experiences (APPEs). INNOVATION: A six-month longitudinal pilot program named the Focused Institutional Longitudinal Experience (FILE) program was developed at two academic medical centers to maximize active participation of the student and minimize the time spent orienting and onboarding students to each APPE experience. A unique component of the FILE program is the longitudinal service project, which involved a medication use evaluation, including a review of published literature and drug policy recommendations to medical center quality committees. ANALYSIS: Student ratings regarding the quality and value of the FILE student experience was compared to the traditional APPE model. Nine quality measures were compared (e.g. amount of opportunity for direct patient care experience, learning, integration into healthcare team, and accountability for patient outcomes) between students from the FILE program to peers completing similar APPEs outside the FILE program. FILE students and APPE preceptors also completed surveys regarding the value of several program aspects. KEY FINDINGS: There was no difference between FILE and non-FILE student self-rated measures of APPE quality, and thus the decision to participate in a longitudinal APPE program should be based on the personal preference of the student. Students in the FILE program agreed or strongly agreed (mean score 4.3) that they felt prepared for post-graduate training at the completion of the program. The potential value that students in a longitudinal program might bring to the site is reinforced by the general agreement by preceptors in the end of year survey that FILE students take less of their time to orient to their service and the trend toward perception that FILE students are more likely to independently participate in patient care activities. NEXT STEPS: To address feedback on preceptor-mentor role and the desire for more interaction with pharmacy residents, students are now paired with a pharmacy resident, and the student and resident work together on the service project with a clinical pharmacist as an advisor. Updated standards of practice clearly delineate the roles and responsibilities of students, residents, and the clinical pharmacist preceptor. Annual surveys of FILE students and preceptors provide necessary feedback to continuously improve the quality of the program.

Abacavir/Dolutegravir/Lamivudine (Triumeq)-Induced Liver Toxicity in a Human Immunodeficiency Virus-Infected Patient.

Drug-induced liver injury related to Triumeq (abacavir/lamivudine/dolutegravir) has not been reported in clinical trials. We report a case of hepatotoxicity related to Triumeq exposure in a human immunodeficiency virus-infected patient. Clinicians should remain aware of the risk for acute and late-onset hepatitis with these agents. Close monitoring is recommended.

Juvenile hormone potentiates ecdysone receptor-dependent transcription in a mammalian cell culture system.

Insect development is guided by the combined actions of ecdysteroids and juvenile hormones (JHs). The transcriptional effects of ecdysteroids are mediated by a protein complex consisting of the ecdysone receptor (EcR) and its heterodimeric partner, Ultraspiracle (USP), but a corresponding JH receptor has not been defined conclusively. Given that the EcR ligand binding domain (LBD) is similar to that of the JH-responsive rat farnesoid-X-activated receptor (FXR), we sought to define experimental conditions under which EcR-dependent transcription could be promoted by JH. Chinese hamster ovary (CHO) cells were transfected with a plasmid carrying an ecdysteroid-inducible reporter gene, a second plasmid expressing one of the three amino-terminal variants of Drosophila EcR or an EcR chimera, and a third plasmid expressing either the mouse retinoid X receptor (RXR), or its insect orthologue, USP. Each of the EcR variants responded to the synthetic ecdysteroid, muristerone A (murA), but a maximal response to 20-hydroxyecdysone (20E) was achieved only for specific EcR combinations with its heterodimeric partner. Notably, the Drosophila EcR isoforms were responsive to 20E only when paired with USP, and only EcRB2 activity was further potentiated by JHIII in the presence of 20E. EcR chimeras that fuse the activator domains from VP16 or the glucocorticoid receptor to the Drosophila EcR DNA-binding and ligand-binding domains were responsive to ecdysteroids. Again, the effects of JHIII and 20E were associated with specific partners of the chimeric EcRs. In all experiments, the LBD of EcR proved to be the prerequisite component for potentiation by JHIII, and in this conformation may resemble the FXR LBD. Our results indicate that EcR responsiveness is influenced by the heterodimeric partner and that both the N-terminal domain of EcR and the particular ecdysteroid affect JHIII potentiation.

Secondary transfer and expression of vanA in Enterococcus faecium derived from a commensal vancomycin-susceptible Enterococcus faecium multi-component food isolate.

We investigated the potential for vancomycin-susceptible Enterococcus (VSE) from multi-component salads to disseminate vanA from four clinical vancomycin-resistant enterococci to 14 streptogramin-resistant enterococci (SRE) of food and animal origin. Strains were selected from a previous study based on the presence of streptogramin susceptibility and/or vanA, vanB, vatD, vatE, agg, cpd, and gelE genes. Transconjugants were isolated on brain heart infusion agar containing vancomycin and selective antibiotics. Thirty-nine matings using a 1:10 donor-recipient ratio for filter and broth methods resulted in transfer of vanA between an agg(+)cpd(+)gelE(+) Enterococcus faecalis donor and an agg(-)cpd(-)gelE(-) streptogramin-susceptible Enterococcus faecium salad recipient at a frequency of 10(-8) per recipient by filter method. Secondary mating of the transconjugant with SR/VSE strains resulted in a two- to four-log-fold greater frequency of transfer. Reverse transcription-polymerase chain reaction revealed vanA RNA products in the transconjugant cultivated in nutrient broth and salad at 37°C in the presence and absence of recipient filtrate. This study demonstrated that native salad VSE disseminated vanA to SRE carrying agg, cpd, and/or gelE. An increase in transfer efficiency resulted from secondary conjugation using the native vancomycin- and streptogramin-susceptible salad strain as the donor.

Antibody-drug conjugates for the treatment of non-Hodgkin's lymphoma: target and linker-drug selection.

Antibody-drug conjugates (ADC), potent cytotoxic drugs covalently linked to antibodies via chemical linkers, provide a means to increase the effectiveness of chemotherapy by targeting the drug to neoplastic cells while reducing side effects. Here, we systematically examine the potential targets and linker-drug combinations that could provide an optimal ADC for the treatment for non-Hodgkin's lymphoma. We identified seven antigens (CD19, CD20, CD21, CD22, CD72, CD79b, and CD180) for potential treatment of non-Hodgkin's lymphoma with ADCs. ADCs with cleavable linkers mediated in vivo efficacy via all these targets; ADCs with uncleavable linkers were only effective when targeted to CD22 and CD79b. In target-independent safety studies in rats, the uncleavable linker ADCs showed reduced toxicity, presumably due to the reduced release of free drug or other toxic metabolites into the circulation. Thus, our data suggest that ADCs with cleavable linkers work on a broad range of targets, and for specific targets, ADCs with uncleavable linkers provide a promising opportunity to improve the therapeutic window for ADCs in humans.

Use of fetal pulse oximetry among high-risk women in labor: a randomized clinical trial.

OBJECTIVE: The purpose of this study was to determine the clinical role of fetal pulse oximetry to reduce cesarean delivery for a nonreassuring fetal heart rate tracing. STUDY DESIGN: Singletons > or =28 weeks were randomized to fetal pulse oximetry plus electronic fetal heart rate monitoring (monitoring + fetal pulse oximetry) or monitoring alone. RESULTS: Overall, 360 women in labor were recruited: 150 cases with monitoring+fetal pulse oximetry and 177 cases with monitoring alone were analyzed. Most demographic, obstetric, and neonatal characteristics were similar. Specifically, the gestational age, cervical dilation, and station of the fetal head were not differential factors. In addition, cesarean delivery for nonreassuring fetal heart rate tracing was not different between the group with monitoring+fetal pulse oximetry (29%) and the group with monitoring alone (32%; relative risk, 0.95; 95% CI, 0.75, 1.22). Likewise, cesarean delivery for arrest disorder was similar between the group with monitoring+fetal pulse oximetry (22%) and the group with monitoring alone (23%; relative risk, 1.05; 95% CI, 0.79, 1.44). However, the decision-to-incision time was shorter for the group with monitoring+fetal pulse oximetry (17.8 +/- 8.2 min) than for the group with monitoring alone (27.7 +/- 13.9 min; P < .0001). CONCLUSION: The use of fetal pulse oximetry with electronic fetal heart rate monitoring does not decrease the rate of cesarean delivery, although it does alter the decision-to-incision time.

Implementation of an automated guideline monitor for secondary prevention of acute myocardial infarction.

Clinical practice guidelines can be used to help improve health care quality, but they are often not optimally implemented. Practice enabling and reinforcing techniques, such as clinical reminders and academic detailing are effective methods for translating guidelines into practice. Following a study showing that we could improve adherence to secondary prevention guidelines for acute myocardial infarction (AMI) using computerized alerts and academic detailing, we implemented an automated monitor to accomplish the same goal in a less labor-intensive manner. This paper describes the implementation of this production application.

Validation of automated event triggers using laboratory values related to two problem-prone drugs.

We used computerized alerts to identify patients with laboratory values that could be related to medication errors associated with digoxin and warfarin. Over a six-week period at two inpatient facilities, we generated 62 laboratory-based alerts for warfarin, and 66 for digoxin. The positive predictive value for these alerts representing a preventable event was 71% and 57% for warfarin and digoxin, respectively.