Sue E. Poynter

Extracellular Hsp72, an endogenous DAMP, is released by virally infected airway epithelial cells and activates neutrophils via Toll-like receptor (TLR)-4

BackgroundNeutrophils play an important role in the pathophysiology of RSV, though RSV does not appear to directly activate neutrophils in the lower airways. Therefore locally produced cytokines or other molecules released by virally-infected airway epithelial cells are likely responsible for recruiting and activating neutrophils. Heat shock proteins (HSPs) are generally regarded as intracellular proteins acting as molecular chaperones; however, HSP72 can also be released from cells, and the implications of this release are not fully understood.MethodsHuman bronchial epithelial cells (16HBE14o-) were infected with RSV and Hsp72 levels were measured by Western blot and ELISA. Tracheal aspirates were obtained from critically ill children infected with RSV and analyzed for Hsp72 levels by ELISA. Primary human neutrophils and differentiated HL-60 cells were cultured with Hsp72 and supernatants analyzed for cytokine production. In some cases, cells were pretreated with polymyxin B prior to treatment with Hsp72. IκBα was assessed by Western blot and EMSAs were performed to determine NF-κB activation. HL-60 cells were pretreated with neutralizing antibody against TLR4 prior to Hsp72 treatment. Neutrophils were harvested from the bone marrow of wild type or TLR4-deficient mice prior to treatment with Hsp72.ResultsInfection of 16HBE14o- with RSV showed an induction of intracellular Hsp72 levels as well as extracellular release of Hsp72. Primary human neutrophils from normal donors and differentiated HL-60 cells treated with increasing concentrations of Hsp72 resulted in increased cytokine (IL-8 and TNFα) production. This effect was independent of the low levels of endotoxin in the Hsp72 preparation. Hsp72 mediated cytokine production via activation of NF-κB translocation and DNA binding. Using bone marrow-derived neutrophils from wild type and TLR4-mutant mice, we showed that Hsp72 directly activates neutrophil-derived cytokine production via the activation of TLR4.ConclusionCollectively these data suggest that extracellular Hsp72 is released from virally infected airway epithelial cells resulting in the recruitment and activation of neutrophils.

Validation of a gene expression-based subclassification strategy for pediatric septic shock

Objective:Septic shock heterogeneity has important implications for clinical trial implementation and patient management. We previously addressed this heterogeneity by identifying three putative subclasses of children with septic shock based exclusively on a 100-gene expression signature. Here we attempted to prospectively validate the existence of these gene expression-based subclasses in a validation cohort. Design:Prospective observational study involving microarray-based bioinformatics. Setting:Multiple pediatric intensive care units in the United States. Patients:Separate derivation (n = 98) and validation (n = 82) cohorts of children with septic shock. Interventions:None other than standard care. Measurements and Main Results:Gene expression mosaics of the 100 class-defining genes were generated for 82 individual patients in the validation cohort. Using computer-based image analysis, patients were classified into one of three subclasses (“A,” “B,” or “C”) based on color and pattern similarity relative to reference mosaics generated from the original derivation cohort. After subclassification, the clinical database was mined for phenotyping. Subclass A patients had higher illness severity relative to subclasses B and C as measured by maximal organ failure, fewer intensive care unit-free days, and a higher Pediatric Risk of Mortality score. Patients in subclass A were characterized by repression of genes corresponding to adaptive immunity and glucocorticoid receptor signaling. Separate subclass assignments were conducted by 21 individual clinicians using visual inspection. The consensus classification of the clinicians had modest agreement with the computer algorithm. Conclusions:We have validated the existence of subclasses of children with septic shock based on a biologically relevant, 100-gene expression signature. The subclasses have relevant clinical differences.

Toward a clinically feasible gene expression-based subclassification strategy for septic shock: proof of concept.

Objective:To develop a clinically feasible stratification strategy for pediatric septic shock, using gene expression mosaics and a 100-gene signature representing the first 24 hrs of admission to the pediatric intensive care unit. Design:Prospective, observational study involving microarray-based bioinformatics. Setting:Multiple pediatric intensive care units in the United States. Patients:Ninety-eight children with septic shock. Interventions:None other than standard care. Measurements and Main Results:Patients were classified into three previously published, genome-wide, expression-based subclasses (subclasses A, B, and C) having clinically relevant phenotypic differences. The class-defining 100-gene signature was depicted for each individual patient, using mosaics generated by the Gene Expression Dynamics Inspector (GEDI). Composite mosaics were generated representing the average expression patterns for each of the three subclasses. Nine individual clinicians served as blinded evaluators. Each evaluator was shown the 98 individual patient mosaics and asked to classify each patient into one of the three subclasses, using the composite mosaics as the reference point. The respective sensitivities, specificities, positive predictive values, and negative predictive values of the subclassification strategy were ≥84% across the three subclasses. The classification strategy also generated positive likelihood ratios of ≥16.8 and negative likelihood ratios of ≤0.2 across the three subclasses. The &kgr; coefficient across all possible interevaluator comparisons was 0.81. Conclusions:We have provided initial evidence (proof of concept) for a clinically feasible and robust stratification strategy for pediatric septic shock based on a 100-gene signature and gene expression mosaics.

Surfactant biology and clinical application

There is strong evidence that alterations in the pulmonary surfactant system play an important role in the pathophysiology of lung disease, including ARDS . Although it is still unclear whether mortality and morbidity of ARDS will be reduced, surfactant replacement therapy has been shown to improve oxygenation, improve lung compliance, and decrease the need for ventilatory support. The critical need for more standardized studies with one type of intratracheal surfactant and uniform measurements of surfactant proteins and phospholipids by BAL is evident. Further studies will also be needed to elucidate the optimal timing and dosage regimen for different disease processes. Some evidence supports the measurements of surfactant protein levels as markers for predicting the onset and outcome of ARDS and perhaps providing a window for early treatment of patients at risk to develop ARDS. Continued investigation into the role of surfactant in the immune regulation of the lung may also provide additional information to support the efficacy of surfactant replacement in lung disease.

Putting the pediatrics milestones into practice: a consensus roadmap and resource analysis.

The Accreditation Council for Graduate Medical Education has partnered with member boards of the American Board of Medical Specialties to initiate the next steps in advancing competency-based assessment in residency programs. This initiative, known as the Milestone Project, is a paradigm shift from traditional assessment efforts and requires all pediatrics residency programs to report individual resident progression along a series of 4 to 5 developmental levels of performance, or milestones, for individual competencies every 6 months beginning in June 2014. The effort required to successfully make this shift is tremendous given the number of training programs, training institutions, and trainees. However, it holds great promise for achieving training outcomes that align with patient needs; developing a valid, reliable, and meaningful way to track residents’ development; and providing trainees with a roadmap for learning. Recognizing the resources needed to implement this new system, the authors, all residency program leaders, provide their consensus view of the components necessary for implementing and sustaining this effort, including resource estimates for completing this work. The authors have identified 4 domains: (1) Program Review and Development of Stakeholders and Participants, (2) Assessment Methods and Validation, (3) Data and Assessment System Development, and (4) Summative Assessment and Feedback. This work can serve as a starting point and framework for collaboration with program, department, and institutional leaders to identify and garner necessary resources and plan for local and national efforts that will ensure successful transition to milestones-based assessment.

Biological activity of truncated C-terminus human heat shock protein 72.

Heat shock protein 72 (Hsp72), a canonical intracellular molecular chaperone, may also function as an extracellular danger signal for the innate immune system. To further delineate the biological role of Hsp72 in the innate immune system, we generated two truncated versions of the full length human Hsp72 (N-terminus Hsp72, amino acids 1-430; and C-terminus Hsp72 amino acids 420-641) and directly compared their ability to activate cells from the macrophage/monocyte lineage. In RAW 264.7 macrophages transfected with a NF-κB-dependent luciferase reporter plasmid, C-terminus Hsp72 was a more potent inducer of NF-κB activity than N-terminus Hsp72, and this effect did not seem to be secondary to endotoxin contamination. C-terminus Hsp72-mediated activation of the NF-κB pathway was corroborated by increased activation of IκB kinase, degradation of IκBα, and increased NF-κB-DNA binding. C-terminus Hsp72 was a more potent inducer of tumor necrosis factor-α (TNFα) expression in RAW 264.7 macrophages and in primary murine peritoneal macrophages from wild-type mice. C-terminus Hsp72 did not induce TNFα expression in primary murine peritoneal macrophages from Toll-like receptor (TLR4) mutant mice, indicating a role for TLR4. In human THP-1 mononuclear cells, C-terminus Hsp72 induced tolerance to subsequent LPS stimulation, whereas N-terminus Hsp72 did not induce tolerance. Finally, control experiments using equimolar amounts of N-terminus or C-terminus Hsp72 demonstrated a higher biological potency for C-terminus Hsp72. These data demonstrate that the ability of human Hsp72 to serve as an activator for cells of the macrophage/monocyte lineage primarily lies in the C-terminus region spanning amino acids 420-641.

The pediatrics milestones assessment pilot: Development of workplace-based assessment content, instruments, and processes

Purpose To report on the development of content and user feedback regarding the assessment process and utility of the workplace-based assessment instruments of the Pediatrics Milestones Assessment Pilot (PMAP). Method One multisource feedback instrument and two structured clinical observation instruments were developed and refined by experts in pediatrics and assessment to provide evidence for nine competencies based on the Pediatrics Milestones (PMs) and chosen to inform residency program faculty decisions about learners’ readiness to serve as pediatric interns in the inpatient setting. During the 2012–2013 PMAP study, 18 U.S. pediatric residency programs enrolled interns and subinterns. Faculty, residents, nurses, and other observers used the instruments to assess learner performance through direct observation during a one-month rotation. At the end of the rotation, data were aggregated for each learner, milestone levels were assigned using a milestone classification form, and feedback was provided to learners. Learners and site leads were surveyed and/or interviewed about their experience as participants. Results Across the sites, 2,338 instruments assessing 239 learners were completed by 630 unique observers. Regarding end-of-rotation feedback, 93% of learners (128/137) agreed the assessments and feedback “helped me understand how those with whom I work perceive my performance,” and 85% (117/137) agreed they were “useful for constructing future goals or identifying a developmental path.” Site leads identified several benefits and challenges to the assessment process. Conclusions PM-based instruments used in workplace-based assessment provide a meaningful and acceptable approach to collecting evidence of learner competency development. Learners valued feedback provided by PM-based assessment.

Validity Evidence From Ratings of Pediatric Interns and Subinterns on a Subset of Pediatric Milestones

Purpose To investigate evidence for validity of faculty members’ pediatric milestone (PM) ratings of interns (first-year residents) and subinterns (fourth-year medical students) on nine subcompetencies related to readiness to serve as a pediatric intern in the inpatient setting. Method The Association of Pediatric Program Directors Longitudinal Educational Assessment Research Network (APPD LEARN) and the National Board of Medical Examiners collaborated to investigate the utility of assessments of the PMs for trainees’ performance. Data from 32 subinterns and 179 interns at 17 programs were collected from July 2012 through April 2013. Observers used several tools to assess learners. At each site, a faculty member used these data to make judgments about the learner’s current developmental milestone in each subcompetency. Linear mixed models were fitted to milestone judgments to examine their relationship with learner’s rank and subcompetency. Results On a 5-point developmental scale, mean milestone levels for interns ranged from 3.20 (for the subcompetency Work effectively as a member of a team) to 3.72 (Humanism) and for subinterns from 2.89 (Organize and prioritize care) to 3.61 (Professionalization). Mean milestone ratings were significantly higher for the Professionalism competency (3.59–3.72) for all trainees compared with Patient Care (2.89–3.24) and Personal and Professional Development (3.33–3.51). Mean intern ratings were significantly higher than mean subintern ratings for all nine subcompetencies except Professionalization, Humanism, and Trustworthiness. Conclusions The PMs had a coherent internal structure and could distinguish between differing levels of trainees, which supports their validation for documenting developmental progression of pediatric trainees.

A novel workplace-based assessment for competency-based decisions and learner feedback

Abstract Background: Increased recognition of the importance of competency-based education and assessment has led to the need for practical and reliable methods to assess relevant skills in the workplace. Methods: A novel milestone-based workplace assessment system was implemented in 15 pediatrics residency programs. The system provided: (1) web-based multisource feedback (MSF) and structured clinical observation (SCO) instruments that could be completed on any computer or mobile device; and (2) monthly feedback reports that included competency-level scores and recommendations for improvement. Results: For the final instruments, an average of five MSF and 3.7 SCO assessment instruments were completed for each of 292 interns; instruments required an average of 4–8 min to complete. Generalizability coefficients >0.80 were attainable with six MSF observations. Users indicated that the new system added value to their existing assessment program; the need to complete the local assessments in addition to the new assessments was identified as a burden of the overall process. Conclusions: Outcomes – including high participation rates and high reliability compared to what has traditionally been found with workplace-based assessment – provide evidence for the validity of scores resulting from this novel competency-based assessment system. The development of this assessment model is generalizable to other specialties.

Thrombocytopenia-associated multi-organ failure caused by diabetic ketoacidosis

Thrombocytopenia‐associated multi‐organ failure (TAMOF) is an increasingly reported entity in the pediatric intensive care unit. The clinical presentation is similar to thrombotic thrombocytopenic purpura, but with no evidence of hemolysis and no schistocytes on peripheral smear. We report a case of TAMOF induced by diabetic ketoacidosis and treated with therapeutic plasma exchange (TPE). Early diagnosis and initiation of TPE significantly decrease the morbidity associated with TAMOF.