Environmental particulate matter (PM) pollutants adversely affect human health, but the molecular basis is poorly understood. The ion channel transient receptor potential vanilloid-1 (TRPV1) has been implicated as a sensor for environmental PM and a mediator of adverse events in the respiratory tract. The objectives of this study were to determine whether TRPV1 can distinguish chemically and physically unique PM that represents important sources of air pollution; to elucidate the molecular basis of TRPV1 activation by PM; and to ascertain the contributions of TRPV1 to human lung cell and mouse lung tissue responses exposed to an insoluble PM agonist, coal fly ash (CFA1). The major findings of this study are that TRPV1 is activated by some, but not all of the prototype PM materials evaluated, with rank-ordered responses of CFA1 Ͼ diesel exhaust PM Ͼ crystalline silica; TRP melastatin-8 is also robustly activated by CFA1, whereas other TRP channels expressed by airway sensory neurons and lung epithelial cells that may also be activated by CFA1, including TRPs ankyrin 1 (A1), canonical 4␣ (C4␣), M2, V2, V3, and V4, were either slightly (TRPA1) or not activated by CFA1; activation of TRPV1 by CFA1 occurs via cell surface interactions between the solid components of CFA1 and specific amino acid residues of TRPV1 that are localized in the putative pore-loop region; and activation of TRPV1 by CFA1 is not exclusive in mouse lungs but represents a pathway by which CFA1 affects the expression of selected genes in lung epithelial cells and airway tissue.

Lentiviral vectors are increasingly utilized in cell and gene therapy applications because they efficiently transduce target cells such as hematopoietic stem cells and T cells. Large-scale production of current Good Manufacturing Practices-grade lentiviral vectors is limited because of the adherent, serum-dependent nature of HEK293T cells used in the manufacturing process. To optimize large-scale clinical-grade lentiviral vector production, we developed an improved production scheme by adapting HEK293T cells to grow in suspension using commercially available and chemically defined serum-free media. Lentiviral vectors with titers equivalent to those of HEK293T cells were produced from SJ293TS cells using optimized transfection conditions that reduced the required amount of plasmid DNA by 50%. Furthermore, purification of SJ293TS-derived lentiviral vectors at 1 L yielded a recovery of 55% ± 14% (n = 138) of transducing units in the starting material, more than a 2-fold increase over historical yields from adherent HEK293T serum-dependent lentiviral vector preparations. SJ293TS cells were stable to produce lentiviral vectors over 4 months of continuous culture. SJ293TS-derived lentiviral vectors efficiently transduced primary hematopoietic stem cells and T cells from healthy donors. Overall, our SJ293TS cell line enables high-titer vector production in serum-free conditions while reducing the amount of input DNA required, resulting in a highly efficient manufacturing option.

Objectives The aims of this study were to develop a population pharmacokinetic model for intravenous paracetamol in preterm and term neonates and to assess the generalizability of the model by testing its predictive performance in an external dataset. Methods Nonlinear mixed-effects models were constructed from paracetamol concentration–time data in NONMEM 7.2. Potential covariates included body weight, gestational age, postnatal age, postmenstrual age, sex, race, total bilirubin, and estimated glomerular filtration rate. An external dataset was used to test the predictive performance of the model through calculation of bias, precision, and normalized prediction distribution errors. Results The model-building dataset included 260 observations from 35 neonates with a mean gestational age of 33.6 weeks [standard deviation (SD) 6.6]. Data were well-described by a one-compartment model with first-order elimination. Weight predicted paracetamol clearance and volume of distribution, which were estimated as 0.348 L/h (5.5 % relative standard error; 30.8 % coefficient of variation) and 2.46 L (3.5 % relative standard error; 14.3 % coefficient of variation), respectively, at the mean subject weight of 2.30 kg. An external evaluation was performed on an independent dataset that included 436 observations from 60 neonates with a mean gestational age of 35.6 weeks (SD 4.3). The median prediction error was 10.1 % [95 % confidence interval (CI) 6.1–14.3] and the median absolute prediction error was 25.3 % (95 % CI 23.1–28.1). Conclusions Weight predicted intravenous paracetamol pharmacokinetics in neonates ranging from extreme preterm to full-term gestational status. External evaluation suggested that these findings should be generalizable to other similar patient populations.

Background: Methamphetamine is an addictive stimulant that can cause many adverse physical, psychological and psychosocial effects. Preliminary evidence shows cannabidiol, a non-intoxicating constituent of the cannabis plant, may have efficacy in treating opioid and nicotine dependence. However, no study has yet examined whether cannabidiol treatment might impact on methamphetamine addiction. Aims: The current study investigated whether cannabidiol administration reduces the motivation to self-administer methamphetamine and relapse to methamphetamine-seeking behavior following abstinence. Methods: Thirty-two male Sprague Dawley rats with implanted jugular vein catheters were initially trained to self-administer methamphetamine via lever press during two-hour sessions on a fixed ratio 1 schedule of reinforcement. Rats in experiment 1 (n=16) then advanced to a progressive ratio reinforcement schedule to examine the effects of cannabidiol (0, 20, 40, and 80 mg/kg intraperitoneal) on motivation to self-administer methamphetamine. Rats in experiment 2 (n=16) were tested for cannabidiol effects on methamphetamine-primed reinstatement following extinction. Results: Cannabidiol (80 mg/kg, but not 40 mg/kg, or 20 mg/kg) reduced the motivation to self-administer methamphetamine and attenuated methamphetamine-primed relapse to methamphetamine-seeking behavior after extinction. Conclusion: This is the first demonstration that cannabidiol can reduce the motivation to seek and consume methamphetamine, and suggests that cannabidiol might be worth trialing as a novel pharmacotherapy for methamphetamine dependence.

IntroductionThe pharmacokinetics of vancomycin are highly variable among neonates, which makes dosing challenging in this population. However, adequate drug exposure is critical, especially when treating methicillin-resistant Staphylococcus aureus (MRSA) infections. Utilization of population pharmacokinetic models and Bayesian methods offers the potential for developing individualized therapeutic approaches. To meet this need, a neonatal vancomycin population pharmacokinetic model was recently published. The current study sought to externally evaluate the predictive performance and generalizability of this model.MethodsA retrospective chart review of neonates who received vancomycin and had ≥1 peak and ≥1 trough concentrations at five Intermountain Healthcare neonatal intensive care units from 2006 to 2013 was performed and served as the external validation cohort. The published population pharmacokinetic model was implemented in NONMEM 7.2 with the structural and variance parameter values set equal to the estimates reported previously. The model was then used to predict the first peak and trough concentration for each neonate in the validation cohort and the model prediction error and absolute prediction error were calculated. Normalized prediction distribution errors (NPDE) were also evaluated.ResultsA total of 243 neonates were studied with a median postmenstrual age of 33 (range: 23–54) weeks and a median weight of 1.6 (range: 0.4–6.8) kg. The model predicted the observed vancomycin concentrations with reasonable precision. For all vancomycin concentrations, the median prediction error was −0.8 (95% CI: −1.4 to −0.4) mg/L and the median absolute prediction error was 3.0 (95% CI: 2.7–3.5) mg/L. No trends in NPDE across weight, postmenstrual age, serum creatinine, or time after dose were observed.ConclusionAn evaluation of a recently published neonatal vancomycin population pharmacokinetic model in a large external dataset supported the predictive performance and generalizability of the model. This model may be useful in evaluating neonatal vancomycin dosing regimens and estimating the extent of drug exposure.Electronic supplementary materialThe online version of this article (doi:10.1007/s40121-015-0067-9) contains supplementary material, which is available to authorized users.

Invasive fungal infections are a significant cause of morbidity and mortality in children. Successful management of these systemic infections requires identification of the causative pathogen, appropriate antifungal selection, and optimisation of its pharmacokinetic and pharmacodynamic properties to maximise its antifungal activity and minimise toxicity and the emergence of resistance. This review highlights salient scientific advancements in paediatric antifungal pharmacotherapies and focuses on pharmacokinetic and pharmacodynamic studies that underpin current clinical decision making. Four classes of drugs are widely used in the treatment of invasive fungal infections in children, including the polyenes, triazoles, pyrimidine analogues and echinocandins. Several lipidic formulations of the polyene amphotericin B have substantially reduced the toxicity associated with the traditional amphotericin B formulation. Monotherapy with the pyrimidine analogue flucytosine rapidly promotes the emergence of resistance and cannot be recommended. However, when used in combination with other antifungal agents, therapeutic drug monitoring of flucytosine has been shown to reduce high peak flucytosine concentrations, which are strongly associated with toxicity. The triazoles feature large inter-individual pharmacokinetic variability, although this pattern is less pronounced with fluconazole. In clinical trials, posaconazole was associated with fewer adverse effects than other members of the triazole family, though both posaconazole and itraconazole display erratic absorption that is influenced by gastric pH and the gastric emptying rate. Limited data suggest that the clinical response to therapy may be improved with higher plasma posaconazole and itraconazole concentrations. For voriconazole, pharmacokinetic studies among children have revealed that children require twice the recommended adult dose to achieve comparable blood concentrations. Voriconazole clearance is also affected by the cytochrome P450 (CYP) 2C19 genotype and hepatic impairment. Therapeutic drug monitoring is recommended as voriconazole pharmacokinetics are highly variable and small dose increases can result in marked changes in plasma concentrations. For the echinocandins, the primary source of pharmacokinetic variability stems from an age-dependent decrease in clearance with increasing age. Consequently, young children require larger doses per kilogram of body weight than older children and adults. Routine therapeutic drug monitoring for the echinocandins is not recommended. The effectiveness of many systemic antifungal agents has been correlated with pharmacodynamic targets in in vitro and in murine models of invasive candidiasis and aspergillosis. Further study is needed to translate these findings into optimal dosing regimens for children and to understand how these agents interact when multiple antifungal agents are used in combination.

Lung inflammation in premature infants contributes to the development of bronchopulmonary dysplasia (BPD), a chronic lung disease with long-term sequelae. Pilot studies administering budesonide suspended in surfactant have found reduced BPD without the apparent adverse effects that occur with systemic dexamethasone therapy. Our objective was to determine budesonide potency, stability, and antiinflammatory effects in human fetal lung. We cultured explants of second-trimester fetal lung with budesonide or dexamethasone and used microscopy, immunoassays, RNA sequencing, liquid chromatography/tandem mass spectrometry, and pulsating bubble surfactometry. Budesonide suppressed secreted chemokines IL-8 and CCL2 (MCP-1) within 4 hours, reaching a 90% decrease at 12 hours, which was fully reversed 72 hours after removal of the steroid. Half-maximal effects occurred at 0.04-0.05 nM, representing a fivefold greater potency than for dexamethasone. Budesonide significantly induced 3.6% and repressed 2.8% of 14,500 sequenced mRNAs by 1.6-to 95-fold, including 119 genes that contribute to the glucocorticoid inflammatory transcriptome; some are known targets of nuclear factor-kB. By global proteomics, 22 secreted inflammatory proteins were hormonally regulated. Two glucocorticoid-regulated genes of interest because of their association with lung disease are CHI3L1 and IL1RL1. Budesonide retained activity in the presence of surfactant and did not alter its surface properties. There was some formation of palmitate-budesonide in lung tissue but no detectable metabolism to inactive 16a-hydroxy prednisolone. We concluded that budesonide is a potent and stable antiinflammatory glucocorticoid in human fetal lung in vitro, supporting a beneficial antiinflammatory response to lung-targeted budesonide:surfactant treatment of infants for the prevention of BPD.

BK virus (BKV) is recognized as a significant cause of renal allograft dysfunction in adults, and there is growing awareness of its importance in the pediatric population. Eighteen pediatric renal transplant recipients and 18 age-matched controls were prospectively studied. Anti-BKV immunoglobulin G (IgG) and IgM titres were assayed in all subjects at entry to the study. Polymerase chain reaction (PCR) for BKV DNA was performed on urine and serum at entry, and prospectively tested again at 4, 8 and 12 months. Mean age +/- s.d. of transplant recipients and controls was 14.6 +/- 3.3 and 13.9 +/- 0.33 yr respectively [not significant (NS)]. Transplant patients were studied at a mean time of 5.6 +/- 4.2 yr post-transplant. 56% of transplant patients and 39% of controls were seropositive (+ve BKV IgG) (NS). Plasma BKV PCR was positive in one transplant patient (who also had positive urine PCR) and in none of the controls. The prevalence of positive urine PCR in transplant patients was greater than in controls (33% vs. 0%, p = 0.02). Positive urine BKV PCR was more commonly found in patients treated with mycophenolate than azathioprine (p = 0.04). We conclude that the prevalence of BKV seropositivity and viral activation in this Australian pediatric renal transplant population is similar to that reported in adult and pediatric populations in other countries. BK viruria was more common in children with greater immunosuppression, suggesting that this group is at higher risk of BKV induced nephropathy.