Extracellular vesicles from vaginal Gardnerella vaginalis and Mobiluncus mulieris contain distinct proteomic cargo and induce inflammatory pathways.

Colonization of the vaginal space with bacteria such as Gardnerella vaginalis and Mobiluncus mulieris is associated with increased risk for STIs, bacterial vaginosis, and preterm birth, while Lactobacillus crispatus is associated with optimal reproductive health. Although host-microbe interactions are hypothesized to contribute to reproductive health and disease, the bacterial mediators that are critical to this response remain unclear. Bacterial extracellular vesicles (bEVs) are proposed to participate in host-microbe communication by providing protection of bacterial cargo, delivery to intracellular targets, and ultimately induction of immune responses from the host. We evaluated the proteome of bEVs produced in vitro from G. vaginalis, M. mulieris, and L. crispatus, identifying specific proteins of immunologic interest. We found that bEVs from each bacterial species internalize within cervical and vaginal epithelial cells, and that epithelial and immune cells express a multi-cytokine response when exposed to bEVs from G. vaginalis and M. mulieris but not L. crispatus. Further, we demonstrate that the inflammatory response induced by G. vaginalis and M. mulieris bEVs is TLR2-specific. Our results provide evidence that vaginal bacteria communicate with host cells through secreted bEVs, revealing a mechanism by which bacteria lead to adverse reproductive outcomes associated with inflammation. Elucidating host-microbe interactions in the cervicovaginal space will provide further insight into the mechanisms contributing to microbiome-mediated adverse outcomes and may reveal new therapeutic targets.

Demonstrating the immunostimulatory and cytokine-augmentation effects of bacterial ghosts on natural killer cells and Caenorhabditis Elegans.

The potential of bacteria-based immunotherapy lies in its ability to inherently enhance immune responses. However, the "liveness" of bacteria poses risks of bacterial escape, nonspecific immuno-stimulation, and ethical concerns, limiting their acceptability in immunotherapy. In this scenario, nonliving empty bacterial-cell envelopes, named bacterial ghosts (BGs), have emerged as immuno-stimulants with the potential to side-step the limitations of live bacterial therapies. This study demonstrates the capability of BGs in modulating the functionality of NK-92 cells and Caenorhabditis elegans (C. elegans), as well as perform as cytokine-therapy adjuvants. BGs were obtained through a pH-driven culture method, and were validated for their structural and chemical integrity via electron microscopy and spectroscopy. In NK-92 cells, BGs have shown significant immuno-stimulation by boosting the gene-expression of perforin, granzyme-B, Fas-L, and interferon-gamma by factors of 3.5-, 1.5-, 12.5-, and 8.6-folds, respectively. Combined BG and IL-12 treatment yielded a notable 10.2-fold increase in interferon-gamma protein expression in 24 h. The BGs also significantly influenced the innate immune response in C. elegans through the upregulation of lysozyme genes viz., ilys-3 (8.8-fold) and lys-2 (3.1-fold). Our investigation into the impact of BGs on natural killer cells and C. elegans highlights its potential as a valid alternative approach for new-age immunotherapy and cytokine augmentation.

Vaginal microbes alter epithelial transcriptomic and epigenomic modifications providing insight into the molecular mechanisms for susceptibility to adverse reproductive outcomes.

The cervicovaginal microbiome is highly associated with women's health with microbial communities dominated by Lactobacillus spp. being considered optimal. Conversely, a lack of lactobacilli and a high abundance of strict and facultative anaerobes including Gardnerella vaginalis, have been associated with adverse reproductive outcomes. However, the molecular pathways modulated by microbe interactions with the cervicovaginal epithelia remain unclear. Using RNA-sequencing, we characterize the in vitro cervicovaginal epithelial transcriptional response to different vaginal bacteria and their culture supernatants. We showed that G. vaginalis upregulated genes were associated with an activated innate immune response including anti-microbial peptides and inflammasome pathways, represented by NLRP3-mediated increases in caspase-1, IL-1ß and cell death. Cervicovaginal epithelial cells exposed to L. crispatus showed limited transcriptomic changes, while exposure to L. crispatus culture supernatants resulted in a shift in the epigenomic landscape of cervical epithelial cells. ATAC-sequencing confirmed epigenetic changes with reduced chromatin accessibility. This study reveals new insight into host-microbe interactions in the lower reproductive tract and suggest potential therapeutic strategies leveraging the vaginal microbiome to improve reproductive health.

A Qualitative Analysis of Student and Preceptor Resiliency Discussions During Introductory Pharmacy Practice Experiences.

OBJECTIVE: The primary objective of this qualitative study was to describe key themes discussed by student pharmacists and preceptors related to recognizing burnout and maintaining wellbeing during an experiential assignment. METHODS: This qualitative study used directed content analysis to examine themes that emerged from discussions between student pharmacists and preceptors as part of a required lifelong learning interview during Introductory Pharmacy Practice Experiences that took place between August 2019 and May 2020. Iterative axial coding was used to develop and redefine codes systematically until consensus was reached and a final codebook was established. All responses were coded using the consensus categories. RESULTS: From the 228 interviews analyzed, 3 overarching themes emerged: causes of burnout, manifestations or symptoms of burnout, and strategies to manage stress and burnout. Preceptors identified the mechanics of the job and feeling extreme pressure or worry as 2 major causes of burnout, which manifest as physical exhaustion, mental exhaustion, and poor work performance. To manage stress, preceptors reported using personal and work-positive coping strategies. CONCLUSION: The major themes derived from the analysis-causes of burnout, manifestations of burnout, and strategies to manage stress-align with what is already known about health care provider wellbeing. Although it remains unknown if this type of wellness intervention is effective at changing student stress management habits, results indicate that the intervention sparked conversations that could assist students in establishing a baseline of knowledge on the topic and supply students with a toolkit of resiliency strategies.

Intrauterine colonization with Gardnerella vaginalis and Mobiluncus mulieris induces maternal inflammation but not preterm birth in a mouse model.

PROBLEM: Preterm birth (PTB) remains a leading cause of childhood mortality. Recent studies demonstrate that the risk of spontaneous PTB (sPTB) is increased in individuals with Lactobacillus-deficient vaginal microbial communities. One proposed mechanism is that vaginal microbes ascend through the cervix, colonize the uterus, and activate inflammatory pathways leading to sPTB. This study assessed whether intrauterine colonization with either Gardnerella vaginalis and Mobiluncus mulieris alone is sufficient to induce maternal-fetal inflammation and induce sPTB. METHOD OF STUDY: C56/B6J mice, on embryonic day 15, received intrauterine inoculation of saline or 108 colony-forming units of G. vaginalis (n = 30), M. mulieris (n = 17), or Lactobacillus crispatus (n = 16). Dams were either monitored for maternal morbidity and sPTB or sacrificed 6 h post-infusion for analysis of bacterial growth and cytokine/chemokine expression in maternal and fetal tissues. RESULTS: Six hours following intrauterine inoculation with G. vaginalis, M. mulieris, or L. crispatus, live bacteria were observed in both blood and amniotic fluid, and a potent immune response was identified in the uterus and maternal serum. In contrast, only a limited immune response was identified in the amniotic fluid and the fetus after intrauterine inoculation. High bacterial load (108 CFU/animal) of G. vaginalis was associated with maternal morbidity and mortality but not sPTB. Intrauterine infusion with L. crispatus or M. mulieris at 108 CFU/animal did not induce sPTB, alter pup viability, litter size, or maternal mortality. CONCLUSIONS: Despite inducing an immune response, intrauterine infusion of live G. vaginalis or M. mulieris is not sufficient to induce sPTB in our mouse model. These results suggest that ascension of common vaginal microbes into the uterine cavity alone is not causative for sPTB.

Nanotherapeutics and the Brain.

Brain disease remains a significant health, social, and economic burden with a high failure rate of translation of therapeutics to the clinic. Nanotherapeutics have represented a promising area of technology investment to improve drug bioavailability and delivery to the brain, with several successes for nanotherapeutic use for central nervous system disease that are currently in the clinic. However, renewed and continued research on the treatment of neurological disorders is critically needed. We explore the challenges of drug delivery to the brain and the ways in which nanotherapeutics can overcome these challenges. We provide a summary and overview of general design principles that can be applied to nanotherapeutics for uptake and penetration in the brain. We next highlight remaining questions that limit the translational potential of nanotherapeutics for application in the clinic. Lastly, we provide recommendations for ongoing preclinical research to improve the overall success of nanotherapeutics against neurological disease.

Surfactants influence polymer nanoparticle fate within the brain.

Drug delivery to the brain is limited by poor penetration of pharmaceutical agents across the blood-brain barrier (BBB), within the brain parenchyma, and into specific cells of interest. Nanotechnology can overcome these barriers, but its ability to do so is dependent on nanoparticle physicochemical properties including surface chemistry. Surface chemistry can be determined by a number of factors, including by the presence of stabilizing surfactant molecules introduced during the formulation process. Nanoparticles coated with poloxamer 188 (F68), poloxamer 407 (F127), and polysorbate 80 (P80) have demonstrated uptake in BBB endothelial cells and enhanced accumulation within the brain. However, the impact of surfactants on nanoparticle fate, and specifically on brain extracellular diffusion or intracellular targeting, must be better understood to design nanotherapeutics to efficiently overcome drug delivery barriers in the brain. Here, we evaluated the effect of the biocompatible and commonly used surfactants cholic acid (CHA), F68, F127, P80, and poly (vinyl alcohol) (PVA) on poly (lactic-co-glycolic acid)-poly (ethylene glycol) (PLGA-PEG) nanoparticle transport to and within the brain. The inclusion of these surfactant molecules decreases diffusive ability through brain tissue, reflecting the surfactant's role in encouraging cellular interaction at short length and time scales. After in vivo administration, PLGA-PEG/P80 nanoparticles demonstrated enhanced penetration across the BBB and subsequent internalization within neurons and microglia. Surfactants incorporated into the formulation of PLGA-PEG nanoparticles therefore represent an important design parameter for controlling nanoparticle fate within the brain.

Formulation and Efficacy of Catalase-Loaded Nanoparticles for the Treatment of Neonatal Hypoxic-Ischemic Encephalopathy.

Neonatal hypoxic-ischemic encephalopathy is the leading cause of permanent brain injury in term newborns and currently has no cure. Catalase, an antioxidant enzyme, is a promising therapeutic due to its ability to scavenge toxic reactive oxygen species and improve tissue oxygen status. However, upon in vivo administration, catalase is subject to a short half-life, rapid proteolytic degradation, immunogenicity, and an inability to penetrate the brain. Polymeric nanoparticles can improve pharmacokinetic properties of therapeutic cargo, although encapsulation of large proteins has been challenging. In this paper, we investigated hydrophobic ion pairing as a technique for increasing the hydrophobicity of catalase and driving its subsequent loading into a poly(lactic-co-glycolic acid)-poly(ethylene glycol) (PLGA-PEG) nanoparticle. We found improved formation of catalase-hydrophobic ion complexes with dextran sulfate (DS) compared to sodium dodecyl sulfate (SDS) or taurocholic acid (TA). Molecular dynamics simulations in a model system demonstrated retention of native protein structure after complexation with DS, but not SDS or TA. Using DS-catalase complexes, we developed catalase-loaded PLGA-PEG nanoparticles and evaluated their efficacy in the Vannucci model of unilateral hypoxic-ischemic brain injury in postnatal day 10 rats. Catalase-loaded nanoparticles retained enzymatic activity for at least 24 h in serum-like conditions, distributed through injured brain tissue, and delivered a significant neuroprotective effect compared to saline and blank nanoparticle controls. These results encourage further investigation of catalase and PLGA-PEG nanoparticle-mediated drug delivery for the treatment of neonatal brain injury.

Governing Transport Principles for Nanotherapeutic Application in the Brain.

Neurological diseases account for a significant portion of the global disease burden. While research efforts have identified potential drugs or drug targets for neurological diseases, most therapeutic platforms are still ineffective at reaching the target location selectively and with high yield. Restricted transport, including passage across the blood-brain barrier, through the brain parenchyma, and into specific cells, is a major cause of ineffective therapeutic delivery. However, nanotechnology is a promising, tailorable platform for overcoming these transport barriers and improving therapeutic delivery to the brain. We provide a transport-oriented analysis of nanotechnology's ability to navigate these transport barriers in the brain. We also provide an opinion on the need for technology development for increasing our capacity to characterize and quantify nanoparticle passage through each transport barrier. Finally, we highlight the importance of incorporating the effect of disease, metabolic state, and regional dependencies to better understand transport of nanotherapeutics in the brain.

Nanoparticle-microglial interaction in the ischemic brain is modulated by injury duration and treatment.

Cerebral ischemia is a major cause of death in both neonates and adults, and currently has no cure. Nanotechnology represents one promising area of therapeutic development for cerebral ischemia due to the ability of nanoparticles to overcome biological barriers in the brain. ex vivo injury models have emerged as a high-throughput alternative that can recapitulate disease processes and enable nanoscale probing of the brain microenvironment. In this study, we used oxygen-glucose deprivation (OGD) to model ischemic injury and studied nanoparticle interaction with microglia, resident immune cells in the brain that are of increasing interest for therapeutic delivery. By measuring cell death and glutathione production, we evaluated the effect of OGD exposure time and treatment with azithromycin (AZ) on slice health. We found a robust injury response with 0.5 hr of OGD exposure and effective treatment after immediate application of AZ. We observed an OGD-induced shift in microglial morphology toward increased heterogeneity and circularity, and a decrease in microglial number, which was reversed after treatment. OGD enhanced diffusion of polystyrene-poly(ethylene glycol) (PS-PEG) nanoparticles, improving transport and ability to reach target cells. While microglial uptake of dendrimers or quantum dots (QDs) was not enhanced after injury, internalization of PS-PEG was significantly increased. For PS-PEG, AZ treatment restored microglial uptake to normal control levels. Our results suggest that different nanoparticle platforms should be carefully screened before application and upon doing so; disease-mediated changes in the brain microenvironment can be leveraged by nanoscale drug delivery devices for enhanced cell interaction.

The Status and Adequacy of Preceptor Orientation and Development Programs in US Pharmacy Schools.

Objective. To identify current preceptor orientation and development programs at US colleges and schools of pharmacy and propose future initiatives for preceptor programs. Methods. An anonymous 28-item survey was administered in January 2017 to 128 experiential education personnel at accredited US schools and colleges of pharmacy. Data from completed survey instruments were tabulated and qualitative responses to open-ended questions were examined using thematic analysis. Results. Eighty-five experiential education administrators participated in the survey (response rate=67%). Most preceptor orientation programs met the majority of requirements as outlined within the Accreditation Council for Pharmacy Education's Standard 20.3, although only 42% of programs mandated preceptor orientation prior to student placement. Two-thirds of respondents offered annual, live preceptor development, and 75% of programs used commercially available online products. Nearly 40% of respondents collaborated with other schools or professional organizations to offer preceptor training. Only 29% of programs had specific requirements for pharmacists to maintain their active preceptor status. Seventy percent of respondents reported spending over $2500 and 39% over $5000 annually on preceptor development. Programs with the highest monetary investment (>$10,000/year) in preceptor development offered multiple venues (live and online) for preceptor training. Programs with significant personnel commitment (≥0.5 FTE devoted to preceptor development) frequently had dedicated site visitors. Conclusion. Preceptor orientation programs at US schools of pharmacy are generally similar, but development programs vary significantly across the Academy. Highly invested programs featured live and online training or site visitors who provided individualized feedback or training. Future studies should explore the cost-effectiveness of program options and their impact on preceptor learning and behaviors.

Pharmacokinetics of nanotechnology-based formulations in pediatric populations.

The development of therapeutics for pediatric use has advanced in the last few decades. However, off-label use of adult medications in pediatrics remains a significant clinical problem. Furthermore, the development of therapeutics for pediatrics is challenged by the lack of pharmacokinetic (PK) data in the pediatric population. To promote the development of therapeutics for pediatrics, the United States Pediatric Formulation Initiative recommended the investigation of nanotechnology-based delivery systems. Therefore, in this review, we provided comprehensive information on the PK of nanotechnology-based formulations from preclinical and clinical studies in pediatrics. Specifically, we discuss the relationship between formulation parameters of nanoformulations and PK of the encapsulated drug in the context of pediatrics. We review nanoformulations that include dendrimers, liposomes, polymeric long-acting injectables (LAIs), nanocrystals, inorganic nanoparticles, polymeric micelles, and protein nanoparticles. In addition, we describe the importance and need of PK modeling and simulation approaches used in predicting PK of nanoformulations for pediatric applications.

Student perceptions of the impact and value of incorporation of reflective writing across a pharmacy curriculum.

INTRODUCTION: To assess student perceptions regarding the impact and value of incorporating written reflection across a pharmacy curriculum. METHODS: Throughout the first three years of the pharmacy curriculum, students use the "What, So What, Now What" format to write 18 reflections within five didactic and six experiential courses. For the 93 students in the class of 2015, individual course evaluation questions measuring perceptions of the impact of reflective writing on their achievement of curricular outcomes were analyzed. Student perceptions of reflective writing's value and impact on their professional development were obtained via a survey administered to the class of 2015 after their third professional year. RESULTS: Students identified reflection (100%) and written reflection (98%) as having some degree of importance to their professional development. However, only 42% of students felt that reflective writing had an appreciable or significant impact on their professional development. Most students indicated that reflective writing had a positive impact on their sensitivity to specific patient populations, ethical behavior, interpersonal skills, and ability to provide patient-centered, interprofessional care. CONCLUSION: This study found that most students found some value in reflective writing, felt that reflective writing positively impacted their self-awareness and professional behaviors, and were able to identify benefits of the reflective writing program.

Determining dominant driving forces affecting controlled protein release from polymeric nanoparticles.

Enzymes play a critical role in many applications in biology and medicine as potential therapeutics. One specific area of interest is enzyme encapsulation in polymer nanostructures, which have applications in drug delivery and catalysis. A detailed understanding of the mechanisms governing protein/polymer interactions is crucial for optimizing the performance of these complex systems for different applications. Using a combined computational and experimental approach, this study aims to quantify the relative importance of molecular and mesoscale driving forces to protein release from polymeric nanoparticles. Classical molecular dynamics (MD) simulations have been performed on bovine serum albumin (BSA) in aqueous solutions with oligomeric surrogates of poly(lactic-co-glycolic acid) copolymer, poly(styrene)-poly(lactic acid) copolymer, and poly(lactic acid). The simulated strength and location of polymer surrogate binding to the surface of BSA have been compared to experimental BSA release rates from nanoparticles formulated with these same polymers. Results indicate that the self-interaction tendencies of the polymer surrogates and other macroscale properties may play governing roles in protein release. Additional MD simulations of BSA in solution with poly(styrene)-acrylate copolymer reveal the possibility of enhanced control over the enzyme encapsulation process by tuning polymer self-interaction. Last, the authors find consistent protein surface binding preferences across simulations performed with polymer surrogates of varying lengths, demonstrating that protein/polymer interactions can be understood in part by studying the interactions and affinity of proteins with small polymer surrogates in solution.

Microstructural alterations of sputum in cystic fibrosis lung disease.

The stasis of mucus secretions in the lungs of cystic fibrosis (CF) patients leads to recurrent infections and pulmonary exacerbations, resulting in decreased survival. Prior studies have assessed the biochemical and biophysical features of airway mucus in individuals with CF. However, these measurements are unable to probe mucus structure on microscopic length scales relevant to key players in the progression of CF-related lung disease, namely, viruses, bacteria, and neutrophils. In this study, we quantitatively determined sputum microstructure based on the diffusion of muco-inert nanoparticle probes in CF sputum and found that a reduction in sputum mesh pore size is characteristic of CF patients with reduced lung function, as indicated by measured FEV1. We also discovered that the effect of ex vivo treatment of CF sputum with rhDNase I (Pulmozyme) on microstructure is dependent upon the time interval between the most recent inhaled rhDNase I treatment and the sample collection. Microstructure of mucus may serve as a marker for the extent of CF lung disease and as a parameter for assessing the effectiveness of mucus-altering agents.

[The Barbie-Matrix: effectiveness of a school-based German program for the primary prevention of anorexia nervosa developed for girls up to the age of 12]. / Die Barbie-Matrix: Wirksamkeit des Programms PriMa zur Primärprävention von Magersucht bei Mädchen ab der 6. Klasse.

More than 25 % of the 12-year-old girls in Thuringia (Germany) show problematic eating behaviour as measured with the Eating-Attitudes-Test (EAT-26D), which corresponds to an increased risk for the development of anorexia nervosa or bulimia nervosa. This was the starting position of a controlled study using a pre-post-design to check the effectiveness of a newly developed German program for the prevention of anorexia nervosa in girls ("PriMa"). 42 Thuringian schools (20 as treatment group) with 1006 girls participated in the pilot study, which lasted from September 2004 to July 2005. Program effectiveness was analysed with mostly standardized questionnaires at three times of measurement (before, after the intervention and at 3 months follow-up) referring to body related self esteem (FBeK), satisfaction with body shape (KEDS), eating behaviour (EAT-26D) and body related attitudes. The program was established in 9 x 90-minute lessons including interactive exercises and discussing especially developed posters that show scenes of a Barbie-doll's life including the reports of a patient suffering from anorexia. Significant improvements on all variables could be reached for the higher risk group (EAT-26D >/= 10 points; = 26,7 %). Mean values in the EAT-26D decreased 5 points at the average which is equivalent with 6.6 % of the EAT-26D range, reflecting a practically significant change effect.