Lee A. Polikoff

Nanoparticles that deliver triplex-forming peptide nucleic acid molecules correct F508del CFTR in airway epithelium

Cystic fibrosis (CF) is a lethal genetic disorder most commonly caused by the F508del mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. It is not readily amenable to gene therapy because of its systemic nature and challenges including in vivo gene delivery and transient gene expression. Here, we use triplex-forming PNA molecules and donor DNA in biodegradable polymer nanoparticles to correct F508del. We confirm modification with sequencing and a functional chloride efflux assay. In vitro correction of chloride efflux occurs in up to 25% of human cells. Deep sequencing reveals negligible off-target effects in partially homologous sites. Intranasal application of nanoparticles in CF mice produces changes in nasal epithelium potential differences consistent with corrected CFTR, with gene correction also detected in lung tissue. This work represents facile genome engineering in vivo with oligonucleotides using a nanoparticle system to achieve clinically relevant levels of gene editing without off-target effects.Cystic fibrosis (CF) is a lethal genetic disorder most commonly caused by the F508del mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. It is not readily amenable to gene therapy because of its systemic nature and challenges including in vivo gene delivery and transient gene expression. Here we use triplex-forming peptide nucleic acids and donor DNA in biodegradable polymer nanoparticles to correct F508del. We confirm modification with sequencing and a functional chloride efflux assay. In vitro correction of chloride efflux occurs in up to 25% of human cells. Deep-sequencing reveals negligible off-target effects in partially homologous sites. Intranasal delivery of nanoparticles in CF mice produces changes in the nasal epithelium potential difference assay, consistent with corrected CFTR function. Also, gene correction is detected in the nasal and lung tissue. This work represents facile genome engineering in vivo with oligonucleotides using a nanoparticle system to achieve clinically relevant levels of gene editing without off-target effects.

Characteristics and Outcomes of Pediatric Rapid Response Teams Before and After Mandatory Triggering by an Elevated Pediatric Early Warning System (PEWS) Score

BACKGROUND The Pediatric Early Warning System (PEWS) was created to identify unstable patients before their deterioration. Rapid response teams (RRTs) were developed to assist with management of such patients. In 2009, our institution mandated the activation of RRTs if a PEWS score was elevated (ie, ≥5). OBJECTIVES The goal of this study was to examine changes in characteristics of RRT calls before and after the implementation of a mandatory hospital policy requiring RRT activation due to an elevated PEWS score. METHODS This study was a retrospective database review, with RRT data from June 2007 to December 2010 examined. A total of 44 RRTs were recorded before mandatory triggering and 69 RRTs afterward in the study period (P = .32). RESULTS Compared with the premandatory group, the mandatory triggering group found that tachycardia was a more frequent trigger for RRTs, with an increase of 26.1% (P = .004). RRTs triggered by a change in mental status/agitation decreased by 22.9% (P = .009). An increase of 15.1% of RRTs required no interventions with mandatory triggering. Nighttime RRTs increased by17.5% (P = .07). There was a trend toward decreased PICU transfers in the mandatory triggering group, with no significant change in code blue calls. CONCLUSIONS A hospital policy of mandating RRT activation based on PEWS scores increased nighttime calls and altered the primary reasons for RRT activation in our center, with no evidence of improvements in patient care. These findings should be interpreted with caution given the relatively rare outcomes the policy is intended to prevent; however, our findings highlight the difficulties inherent in evaluating methods to improve pediatric patient safety.

The effect of laser iridotomy on the anterior segment anatomy of patients with plateau iris configuration.

Purpose:To determine if laser iridotomy altered the anterior segment anatomy of patients with plateau iris configuration. Methods:Twenty eyes of 9 female and 1 male patients were imaged using an ultrasound biomicroscope within 19 weeks before and 52 weeks after laser iridotomy. Measurements obtained included the anterior chamber depth (ACD), trabecular-ciliary process distance (TCPD), iris thickness (IT), angle opening distance at 500 micrometers (AOD), iridozonular distance (IZD), and trabecular-iris angle (TIA). Comparisons of the pre- and post- iridotomy measurements were made using a two-tailed paired t test. Results:Laser iridotomy elicited no statistically significant change in ACD, TCPD, IT, AOD, or TIA. However, IZD was decreased (P < 0.05) in both eyes after laser iridotomy. Configuration of the irides was flat before and after laser iridotomies. Conclusion:This study suggests that laser iridotomy did not alter anterior segment anatomy, probably because of the fixed anterior insertion of the iris and ciliary body in plateau iris configuration. The decrease in IZD distance may be the result of a small posterior movement of the iris due to a reduction in relative pupillary block, secondary to laser iridotomy. The small reduction in relative papillary block in plateau iris configuration does not alter the width of the anterior chamber angle as measured by AOD and TIA.

Is intraocular pressure in the early postoperative period predictive of antimetabolite-augmented filtration surgery success?

Purpose:To determine whether intraocular pressure (IOP) in the early postoperative period after trabeculectomy or combined phacoemulsification-trabeculectomy, augmented with antimetabolite, correlates with IOP at one year in surgeries considered to be successful at that time point. Design:Retrospective case series. Methods:A chart review of antimetabolite-augmented surgical procedures done by DJG and JBS between January 1994 and November 2000 identified 82 primary or secondary trabeculectomies and 53 combined phacoemulsification-trabeculectomies with at least one year of follow-up. The success rate for each surgical subgroup was calculated and IOP on postoperative days (POD ± SD) 1, 7 (±2), 30 (±5), 90 (±10), and 180 (±20) was correlated with IOP at one year (POY 1, between month 12 and 15) using linear regression. IOP at each time point was compared among eyes that achieved success at one year with and without the use of IOP-lowering agents. Results:Of the 82 eyes having undergone antimetabolite-augmented trabeculectomies and the 53 eyes having undergone combined surgeries with at least one year of follow-up, the surgical success rates at POY 1 were 87.8% (72 of 82 eyes) and 92.5% (49 of 53 eyes). Of these, 42 eyes (58.3%) from 39 patients in the trabeculectomy group and 27 eyes (55.1%) from 24 patients in the combined surgery group did not require glaucoma medications at one year postsurgically, and were considered complete surgical successes. Mean preoperative IOP mm Hg ± SD was 26.0 ± 8.5 for the trabeculectomy group and 18.2 ± 4.5 for the phaco-trabeculectomy group. Postoperative IOP at POD 1, POD 7, POD 30, POD 90, POD 180, and POY 1 respectively for the eyes undergoing trabeculectomy were 13.9 ± 10.4, 9.5 ± 6.2, 12.0 ± 5.5, 12.0 ± 5.2, 12.8 ± 5.9, and 12.1 ± 4.3, and for the combined surgery group were 20.8 ± 12.5, 9.7 ± 5.7, 12.2 ± 5.4, 11.1 ± 3.4, 11.6 ± 4.6, and 10.3 ± 4.3. Intraocular pressure on postoperative day one correlated poorly with intraocular pressure at POY 1 for the trabeculectomy group (R2 = 0.0788), and not at all for the combined procedures group (R2 = 0.018). The correlation was slightly better for intraocular pressure at postoperative day 90 for the trabeculectomy group (R2 = 0.546), and at postoperative day 180 for the combined group (R2 = 0.37), but still rather low. Eyes requiring glaucoma medication use at POY 1 in the trabeculectomy group had higher (P < 0.009) intraocular pressure at POD 30 and at all subsequent visits than eyes not requiring these medications. Eyes requiring glaucoma medication use at POY 1 in the phaco-trabeculectomy group had higher (P < 0.0025) intraocular pressure at POD 30, POD 180, and POY 1 than eyes not requiring these medications. Conclusion:Intraocular pressure in the early postoperative period correlates very poorly with intraocular pressure one year after successful antimetabolite-augmented trabeculectomy or combined cataract extraction and trabeculectomy. Starting one month after glaucoma surgery, intraocular pressure is substantially lower in eyes that will ultimately not require the use of ocular hypotensive agents to achieve clinical success one year postoperatively.

Beta-adrenergic stimulation of Na+–K+–2Cl− cotransport activity in the rabbit lens

Experimental maneuvers known to increase cellular cAMP levels evoked a stimulation in the K(+) influx across the anterior surfaces of isolated rabbit lenses, as measured by 86Rb(+) uptake. For this, the lenses were mounted in a modified Ussing-type chamber and exposed to the radiolabel under short-circuit conditions. The enhanced, cAMP-elicited flux was attributed to the basolateral Na(+)-K(+)-2Cl(-) cotransporter given its preclusion by bumetanide, a highly selective inhibitor of this symport, and the ineffectiveness of ouabain in mitigating the stimulation. The ouabain- plus bumetanide-insensitive K(+) uptake, which is about 10% of the total influx and represents passive entry of the radiolabel, was not affected by cAMP-elevating conditions. Forskolin, an activator of adenylyl cyclase; epinephrine, a non-selective adrenergic agonist; and the beta-selective agents, isoproterenol and terbutaline, were among the drugs used to elicit the increase in bumetanide-sensitive K(+) inflow. In experiments with isoproterenol, the stimulated influx evoked by the agonist was inhibited in lenses simultaneously exposed to propranolol. Other observations included that the stimulation of bumetanide-sensitive K(+) influx with forskolin was eliminated in lenses pretreated with the protein kinase inhibitors, staurosporine or H-89. However, these drugs were ineffective in preventing the increased influx produced by calyculin A, a phosphatase inhibitor, suggesting modulation of the cotransporter by at least two independent pathways. The cAMP-generating stimuli also produced an inhibition of the short-circuit current across the lens and an increase in translens resistance. These latter effects suggest that cAMP elevation also evokes an inhibition in an epithelial conductance(s) simultaneously to the stimulation of the cotransporter. As such, this study provides the first indication for the regulation of lens transport by adrenoceptors, presumably of the beta-2 subtype.

Additivity of pilocarpine to bimatoprost in ocular hypertension and early glaucoma.

Purpose:To determine if the intraocular pressure (IOP) effect of pilocarpine at various concentrations is additive to that of bimatoprost and to assess the tolerability of this combination. Methods:This was a randomized, prospective trial of patients with IOP > 21 mm Hg following appropriate medication washout. For all visits IOP was measured at 9:00 AM and 11:00 AM. Following baseline visit (#1), bimatoprost 0.03% was instilled qhs OU through visit 6. Following visits 2, 3, and 4 pilocarpine (2%, 4%, 6%) was instilled qid in one randomly selected eye. Pilocarpine was discontinued after visit 5 and bimatoprost after visit 6. Two-tailed, paired t test was used to compare treated and contralateral eyes for their IOP, IOP change, percentage IOP change from baseline, and to compare IOP in the same eye at 9:00 AM and 11:00 AM (before and after pilocarpine administration). IOPs using bimatoprost alone or in combination with various pilocarpine concentrations were compared using single variant Analysis of Variance (ANOVA). Results:Seventeen patients were enrolled and 13 patients completed the study. Bimatoprost reduced IOP 28.7% to 30.5% (P < 0.0001) from baseline to visit 2. IOPs in eyes treated with bimatoprost alone or with bimatoprost and various pilocarpine concentrations were similar (P > 0.81, ANOVA). The IOP (P > 0.17) and percentage IOP change from baseline (P > 0.10) was similar in treated and contralateral eyes with all three strengths of pilocarpine. IOP values at 9:00 AM and 11:00 AM, before and after pilocarpine administration, were similar (P > 0.22). Conclusion:Bimatoprost alone reduces IOP substantially. Pilocarpine added to bimatoprost at concentrations of 2%, 4%, or 6% was neither additive nor antagonistic to the ocular hypotensive efficacy of bimatoprost.

Relationship Between Adverse Tracheal Intubation Associated Events and PICU Outcomes

Objective: Tracheal intubation in PICUs is a common procedure often associated with adverse events. The aim of this study is to evaluate the association between immediate events such as tracheal intubation associated events or desaturation and ICU outcomes: length of stay, duration of mechanical ventilation, and mortality. Study Design: Prospective cohort study with 35 PICUs using a multicenter tracheal intubation quality improvement database (National Emergency Airway Registry for Children: NEAR4KIDS) from January 2013 to June 2015. Desaturation defined as Spo2 less than 80%. Setting: PICUs participating in NEAR4KIDS. Patients: All patients less than18 years of age undergoing primary tracheal intubations with ICU outcome data were analyzed. Measurements and Main Results: Five thousand five hundred four tracheal intubation encounters with median 108 (interquartile range, 58–229) tracheal intubations per site. At least one tracheal intubation associated event was reported in 892 (16%), with 364 (6.6%) severe tracheal intubation associated events. Infants had a higher frequency of tracheal intubation associated event or desaturation than older patients (48% infants vs 34% for 1–7 yr and 18% for 8–17 yr). In univariate analysis, the occurrence of tracheal intubation associated event or desaturation was associated with a longer mechanical ventilation (5 vs 3 d; p < 0.001) and longer PICU stay (14 vs 11 d; p < 0.001) but not with PICU mortality. The occurrence of severe tracheal intubation associated events was associated with longer mechanical ventilation (5 vs 4 d; p < 0.003), longer PICU stay (15 vs 12 d; p < 0.035), and PICU mortality (19.9% vs 9.6%; p < 0.0001). In multivariable analyses, the occurrence of tracheal intubation associated event or desaturation was significantly associated with longer mechanical ventilation (+12%; 95% CI, 4–21%; p = 0.004), and severe tracheal intubation associated events were independently associated with increased PICU mortality (OR = 1.80; 95% CI, 1.24–2.60; p = 0.002), after adjusted for patient confounders. Conclusions: Adverse tracheal intubation associated events and desaturations are common and associated with longer mechanical ventilation in critically ill children. Severe tracheal intubation associated events are associated with higher ICU mortality. Potential interventions to decrease tracheal intubation associated events and oxygen desaturation, such as tracheal intubation checklist, use of apneic oxygenation, and video laryngoscopy, may need to be considered to improve ICU outcomes.

Venous thromboembolism in critically ill children.

Purpose of review To review the current literature on venous thromboembolism (VTE) in critically ill children. Recent findings There is an increasing concern for VTE and its complications in critically ill children. Critically ill children are at increased risk of thromboembolism because of the treatment that they are receiving and their underlying condition. A complex relationship exists between thrombosis and infection. A thrombus is a nidus for infection, while infection increases the risk of thrombosis. Pediatric-specific guidelines for the prevention and treatment of thromboembolism are lacking. Current guidelines are based on the data from adults. Novel anticoagulants are now available for use in adults. Studies are ongoing to determine their safety in children. Risk assessment tools have recently been developed to determine the risk of thromboembolism in critically ill children. Certain molecules are associated with thromboembolism in adults. Summary Pediatric critical care practitioners should be cognizant of the importance of VTE in critically ill children to allow early identification and treatment. Adequately powered clinical trials are critically needed to generate evidence that will guide the treatment and prevention of thromboembolism in critically ill children. Risk assessment tools that incorporate biomarkers may improve our ability to predict the occurrence of thromboembolism in critically ill children.

End-of-life care in the pediatric ICU

Purpose of review Pediatric ICUs frequently provide end-of-life (EOL) care to children. Our understanding of how EOL care is delivered to children and what constitutes effective care for dying children and their families in the ICU setting continues to evolve. This review identifies recent work describing events related to the death of a child in the ICU as well as interventional efforts to improve family and provider support. Recent findings Pediatric ICUs (PICUs) often provide EOL care to children who die in the developed world. Areas of active investigation include identifying effective communication techniques, meeting the needs of patients and parents, and providing support to care providers. Summary PICU practitioners are developing flexible and novel approaches to pediatric EOL care in the ICU setting.

Beta-adrenergic inhibition of rabbit lens anterior-surface K(+) conductance.

Purpose. To characterize the effects of cAMP-elevating stimuli on the rabbit translens electrical parameters and examine the distribution of beta adrenoceptors about the epithelial surface. Methods. The electrophysiological experiments encompassed the isolation of lenses within a vertically arranged, Ussing-type chamber under short-circuit conditions, an approach that allowed for measurements of short-circuit current (I sc) across, in separate experiments, discrete surface regions. Epithelial beta receptors were localized by immunofluorescent labeling of lens cryosections primarily exposed to a polyclonal antibody against human ß 2 -adrenoceptors. Reverse transcription – polymerase chain reaction (RT-PCR) was used to generate cDNA (using specific primers based upon the sequence of the previously cloned human ß 2 receptor) from rabbit lens RNA extracted from mechanically sequestered anterior and equatorial epithelial cells. Results. Asymmetrical I sc reductions with increases in translens resistance were elicited with epinephrine, isoproterenol, terbutaline, forskolin, and a lipid-permeable cAMP analogue. Electrical changes were recorded across the anterior aspect and not observed when the above compounds were applied to solutions bathing the equatorial and posterior surfaces. Immunohistochemical observations indicated the expression of beta receptors from the anterior epithelium to the equatorial region. RT-PCR yielded cDNA of expected basepair length for the apparent fragment of the ß 2 -adrenoceptor, which exhibited a sequence homology 90% identical with its human equivalent in both the anterior and equatorial epithelia. Conclusions. The cAMP-sensitive conductance(s) appear limited to the anterior epithelium and undetectable equatorially. The asymmetrical I sc responses do not seem to arise from a spatial heterogeneity in epithelial receptor expression.