A 3D in vitro assay to study combined immune cell infiltration and cytotoxicity.

Immune cell-mediated killing of cancer cells in a solid tumor is prefaced by a multi-step infiltration cascade of invasion, directed migration, and cytotoxic activities. In particular, immune cells must invade and migrate through a series of different extracellular matrix (ECM) boundaries and domains before reaching and killing their target tumor cells. These infiltration events are a central challenge to the clinical success of CAR T cells against solid tumors. The current standard in vitro cell killing assays measure cell cytotoxicity in an obstacle-free, two-dimensional (2D) microenvironment, which precludes the study of 3D immune cell-ECM interactions. Here, we present a 3D combined infiltration/cytotoxicity assay based on an oil-in-water microtechnology. This assay measures stromal invasion following extravasation, migration through the stromal matrix, and invasion of the solid tumor in addition to cell killing. We compare this 3D cytotoxicity assay to the benchmark 2D assay through tumor assembloid cocultures with immune cells and engineered immune cells. This assay is amenable to an array of imaging techniques, which allows direct observation and quantification of each stage of infiltration in different immune and oncological contexts. We establish the 3D infiltration/cytotoxicity assay as an important tool for the mechanistic study of immune cell interactions with the tumor microenvironment.

Precision-engineered biomimetics: the human fallopian tube.

The fallopian tube has an essential role in several physiological and pathological processes from pregnancy to ovarian cancer. However, there are no biologically relevant models to study its pathophysiology. The state-of-the-art organoid model has been compared to two-dimensional tissue sections and molecularly assessed providing only cursory analyses of the model's accuracy. We developed a novel multi-compartment organoid model of the human fallopian tube that was meticulously tuned to reflect the compartmentalization and heterogeneity of the tissue's composition. We validated this organoid's molecular expression patterns, cilia-driven transport function, and structural accuracy through a highly iterative platform wherein organoids are compared to a three-dimensional, single-cell resolution reference map of a healthy, transplantation-quality human fallopian tube. This organoid model was precision-engineered to match the human microanatomy. One sentence summary: Tunable organoid modeling and CODA architectural quantification in tandem help design a tissue-validated organoid model.

Association Between Centralization and Outcome for Children Admitted to Intensive Care in Australia and New Zealand: A Population-Based Cohort Study.

OBJECTIVES: To describe regional differences and change over time in the degree of centralization of pediatric intensive care in Australia and New Zealand (ANZ) and to compare the characteristics and ICU mortality of children admitted to specialist PICUs and general ICUs (GICUs). DESIGN: A retrospective cohort study using registry data for two epochs of ICU admissions, 2003-2005 and 2016-2018. SETTING: Population-based study in ANZ. PATIENTS: A total of 43,256 admissions of children aged younger than 16 years admitted to an ICU in ANZ were included. Infants aged younger than 28 days without cardiac conditions were excluded. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: The primary outcome was risk-adjusted ICU mortality. Logistic regression was used to investigate the association of mortality with the exposure to ICU type, epoch, and their interaction. Compared with children admitted to GICUs, children admitted to PICUs were younger (median 25 vs 47 mo; p < 0.01) and stayed longer in ICU (median 1.6 vs 1.0 d; p < 0.01). For the study overall, 93% of admissions in Australia were to PICUs whereas in New Zealand only 63% of admissions were to PICUs. The adjusted odds of death in epoch 2 relative to epoch 1 decreased (adjusted odds ratio [AOR], 0.50; 95% CI, 0.42-0.59). There was an interaction between unit type and epoch with increased odds of death associated with care in a GICU in epoch 2 (AOR, 1.63; 95% CI, 1.05-2.53 for all admissions; 1.73, CI, 1.002-3.00 for high-risk admissions). CONCLUSIONS: Risk-adjusted mortality of children admitted to specialist PICUs decreased over a study period of 14 years; however, a similar association between time and outcome was not observed in high-risk children admitted to GICUs. The results support the continued use of a centralized model of delivering intensive care for critically ill children.

Factors associated with admission of children to an intensive care unit and readmission to hospital within 28 days of discharge: A population-based study.

AIM: Hospital readmissions within 28 days are an important performance measurement of quality and safety of health care. The aims of this study were to examine the rates, trends and characteristics of paediatric intensive care unit admissions, and factors associated with readmissions to hospital within 28 days of discharge. METHODS: This retrospective, population-based record linkage study included all children ≥28 days and <16 years old admitted to an intensive care unit (ICU) in a New South Wales (NSW) public hospital from 2004 to 2013. Data were sourced from the NSW Admitted Patients Data Collection and the NSW Registry of Births, Deaths and Marriages, Death Registration. RESULTS: We identified 21 200 ICU admissions involving 17 130 children. Admissions increased by 24% over the study period with the greatest increase attributed to respiratory and musculoskeletal conditions. A higher proportion of children were <5 years, male, lived in major cities, were publicly insured and had chronic conditions. The median length of ICU stay was 42 h and overall hospital stay was 7 days. There were 905 deaths, two-thirds during the index admission with the leading causes being injuries, cancer and infections. Twenty-three per cent of ICU admissions were readmitted to hospital within 28 days of discharge. Associated independent factors were younger age, longer index hospital stay and emergency index admission. Children with chronic conditions of cancer and genitourinary disorders were more likely to be readmitted. CONCLUSIONS: Identification of complex chronic conditions, consideration of long-term health planning and interventions intended to reduce readmission is warranted in order to reduce the burden to families and the health-care system.

The Effect of Imputation of PaO2/FIO2 From SpO2/FIO2 on the Performance of the Pediatric Index of Mortality 3.

OBJECTIVES: To investigate if the performance of Pediatric Index of Mortality 3 is improved by including imputed values for the PaO2/FIO2 ratio where measurements of PaO2 or FIO2 are missing. DESIGN: A prospective observational study. SETTING: A bi-national pediatric intensive care registry. PATIENTS: The records of 37,983 admissions of children less than 16 years old admitted to 19 ICUs. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Seven published equations describing an association between PaO2/FIO2 and oxygen saturation measured by pulse oximetry (SpO2)/FIO2 were used to derive an alternative variable d100 × FIO2/PaO2 for the Pediatric Index of Mortality 3 variable 100 × FIO2/PaO2. Six equations exclude SpO2/FIO2 values if SpO2 is greater than 96-98%. 100 × FIO2/PaO2 was missing in 72% of patient records primarily due to missing PaO2, d100 × FIO2/PaO2 was missing in 71% of patient records if values of SpO2greater than 97% were excluded or in 17% of patient records if all measurements of SpO2 were included. Univariable analysis supported the inclusion of SpO2 values greater than 97%. Compared to the standard Pediatric Index of Mortality 3 model, two alternative models imputing 100 × FIO2/PaO2 from d100 × FIO2/PaO2 only if 100 × FIO2/PaO2 was missing, or using d100 × FIO2/PaO2 values exclusively, resulted in a small but statistically significant improvements in discrimination of Pediatric Index of Mortality 3 (area under the receiver operator curve 0.9068 [0. 8965-0. 9171]; 0.9083 [0.8981-0.9184]; 0.9087 [0.8987-0.9188], respectively). CONCLUSIONS: Imputation of the PaO2/FIO2 ratio in cases where arterial sampling was not performed resulted in a large reduction in the rate of missing data if all values of SpO2 were included. The imputation technique improved the discrimination of Pediatric Index of Mortality 3; however, the magnitude of the increment in overall model performance was small. A possible benefit of the approach is reducing the potential for bias resulting from variation in practice for invasive monitoring of oxygenation.

Humidified high-flow nasal cannula oxygen in bronchiolitis reduces need for invasive ventilation but not intensive care admission.

AIM: To describe the changes to paediatric intensive care unit (PICU) admission patterns and ventilation requirements for children with bronchiolitis following the introduction of humidified high-flow nasal cannula oxygen outside the PICU. METHODS: Retrospective study comparing patients <24 months of age with a discharge diagnosis of bronchiolitis admitted to the PICU. A comparison was made between those before humidified high-flow nasal cannula oxygen use (year 2008) to those immediately following the introduction of humidified high-flow nasal cannula oxygen use (year 2011) and those following further consolidation of humidified high-flow nasal cannula oxygen use outside the PICU (year 2013). RESULTS: Humidified high-flow nasal cannula oxygen use up to 1 L/kg/min in the hospital did not reduce PICU admission. Intubation rates were reduced from 22.2% in 2008 to 7.8% in 2013. There was a non-significant trend towards decreased length of stay in the PICU while hospital length of stay showed a significant decrease following the introduction of humidified high-flow nasal cannula oxygen. Age <6 months and respiratory syncytial virus bronchiolitis were associated with an increased chance of failing humidified high-flow nasal cannula oxygen therapy. CONCLUSION: Humidified high-flow nasal cannula oxygen utilised outside of the PICU in our institution for children with bronchiolitis did not reduce admission rates or length of stay to the PICU but was associated with a decreasing need for invasive ventilation and reduced hospital length of stay.

Contribution of hydrogen bonds to protein stability.

Our goal was to gain a better understanding of the contribution of the burial of polar groups and their hydrogen bonds to the conformational stability of proteins. We measured the change in stability, Δ(ΔG), for a series of hydrogen bonding mutants in four proteins: villin headpiece subdomain (VHP) containing 36 residues, a surface protein from Borrelia burgdorferi (VlsE) containing 341 residues, and two proteins previously studied in our laboratory, ribonucleases Sa (RNase Sa) and T1 (RNase T1). Crystal structures were determined for three of the hydrogen bonding mutants of RNase Sa: S24A, Y51F, and T95A. The structures are very similar to wild type RNase Sa and the hydrogen bonding partners form intermolecular hydrogen bonds to water in all three mutants. We compare our results with previous studies of similar mutants in other proteins and reach the following conclusions. (1) Hydrogen bonds contribute favorably to protein stability. (2) The contribution of hydrogen bonds to protein stability is strongly context dependent. (3) Hydrogen bonds by side chains and peptide groups make similar contributions to protein stability. (4) Polar group burial can make a favorable contribution to protein stability even if the polar groups are not hydrogen bonded. (5) The contribution of hydrogen bonds to protein stability is similar for VHP, a small protein, and VlsE, a large protein.

The capability of static and dynamic features to distinguish competent from genuinely expert practitioners in pediatric diagnosis.

OBJECTIVE: The authors describe the development of a new, more objective method of distinguishing experienced competent nonexpert from expert practitioners within pediatric intensive care. BACKGROUND: Expert performance involves the acquisition and use of refined feature-event associations (cues) in the operational environment. Competent non-experts, although experienced, possess rudimentary cue associations in memory. Thus, they cannot respond as efficiently or as reliably as their expert counterparts, particularly when key diagnostic information is unavailable, such as that provided by dynamic cues. METHOD: This study involved the application of four distinct tasks in which the use of relevant cues could be expected to increase both the accuracy and the efficiency of diagnostic performance. These tasks included both static and dynamic stimuli that were varied systematically. A total of 50 experienced pediatric intensive staff took part in the study. RESULTS: The sample clustered into two levels across the tasks: Participants who performed at a consistently high level throughout the four tasks were labeled experts, and participants who performed at a lower level throughout the tasks were labeled competent nonexperts. The groups differed in their responses to the diagnostic scenarios presented in two of the tasks and their ability to maintain performance in the absence of dynamic features. CONCLUSION: Experienced pediatricians can be decomposed into two groups on the basis of their capacity to acquire and use cues; these groups differ in their diagnostic accuracy and in their ability to maintain performance in the absence of dynamic features. APPLICATION: The tasks may be used to identify practitioners who are failing to acquire expertise at a rate consistent with their experience, position, or training. This information may be used to guide targeted training efforts.

Ventilator-associated pneumonia in a tertiary paediatric intensive care unit: a 1-year prospective observational study.

OBJECTIVES: To determine the incidence, risk factors and impact of ventilator-associated pneumonia (VAP) in a mixed tertiary paediatric intensive care unit. DESIGN: Prospective observational study. METHODS: Patients in the intensive care unit who were mechanically ventilated for more than 48 hours were assessed daily, according to criteria for a diagnosis of VAP. Potential risk factors for VAP, if present, were documented. RESULTS: Of 692 invasively ventilated patients, 269 (38.9%) were ventilated for > 48 hours and met no exclusion criteria. Eighteen (6.7%) patients had episodes of VAP, and the VAP incidence density was 7.02 per 1000 intubation days. The mean admission Paediatric Index of Mortality 2 risk of death was similar in patients with and without VAP (0.084 v 0.056; P =0.8). Patients with VAP (compared with patients without VAP) had a longer median duration of ICU stay, (19.35 v 7.35 days; P < 0.001), duration of ventilation (11.99 v 4.92 days; P=0.024) and duration of hospital stay (35.5 v 20 days; P < 0.001). Univariate analysis showed that reintubation, absence of tube feeding and absence of stress ulcer prophylaxis were risk factors for VAP. While backward selection removed reintubation as a positive predictor during multivariate analysis, tube feeds (hazard ratio (HR), 0.27; 95% CI, 0.09-0.85; P = 0.02) and stress ulcer prophylaxis (HR, 0.29; 95% CI, 0.11-0.76; P = 0.01) were independently associated with reduced VAP incidence. CONCLUSIONS: VAP in children is associated with significant morbidity and increased length of hospital stay. Enteral feeding and stress ulcer prophylaxis while intubated are associated with lower VAP hazards.

Traumatic bleeding at birth treated with Factor VII.

We report 36 week gestation twins born following a traumatic delivery. Twin 2 had profuse haemorrhage where haemostasis was achieved with recombinant Factor VIIa (rFVIIa - NovoSeven;Novo Nordisk A/S, Bagsvaerd, Denmark).

Peri-operative management of paediatric patients undergoing cardiac surgery--focus on respiratory aspects of care.

Children requiring cardiac surgery present particular challenges in peri-operative respiratory management. The wide variety of conditions and operations and their varied impact on respiratory function makes dialogue with related medical staff essential. In most circumstances, cardiac performance is the main determinant of respiratory outcomes. Changing cardiologic and surgical approaches have combined to diminish the severity and frequency of pulmonary hypertensive issues and new treatment modalities are simplifying the intensive care approach. Patients with Down's syndrome and 22q11 deletion syndrome present particular issues related to anatomy, physiology and respiratory function. Certain conditions, including tetralogy of Fallot and cavopulmonary connections, present unique circumstances where respiratory management, sometimes including extubation, may assist in optimisation of cardiac performance. These and other conditions highlight the complexities of cardiopulmonary interactions. Cardiac performance remains the principal determinant of outcome after paediatric cardiac surgery and has the biggest impact on respiratory function.

Hydrogen bonding increases packing density in the protein interior.

The contribution of hydrogen bonds and the burial of polar groups to protein stability is a controversial subject. Theoretical studies suggest that burying polar groups in the protein interior makes an unfavorable contribution to the stability, but experimental studies show that burying polar groups, especially those that are hydrogen bonded, contributes favorably to protein stability. Understanding the factors that are not properly accounted for by the theoretical models would improve the models so that they more accurately describe experimental results. It has been suggested that hydrogen bonds may contribute to protein stability, in part, by increasing packing density in the protein interior, and thereby increasing the contribution of van der Waals interactions to protein stability. To investigate the influence of hydrogen bonds on packing density, we analyzed 687 crystal structures and determined the volume of buried polar groups as a function of their extent of hydrogen bonding. Our findings show that peptide groups and polar side chains that form hydrogen bonds occupy a smaller volume than the same groups when they do not form hydrogen bonds. For example, peptide groups in which both polar groups are hydrogen bonded occupy a volume, on average, 5.2 A3 less than a peptide group that is not hydrogen bonded.

Recombinant activated factor VII in paediatric cardiac surgery.

OBJECTIVE: To review the use of recombinant activated factor VII in paediatric cardiac surgery. DESIGN: Retrospective chart review. SETTING: Paediatric intensive care unit in a stand-alone university-affiliated children's hospital. PATIENTS AND PARTICIPANTS: Cardiac surgical patients who received recombinant activated factor VII (rFVIIa, NovoSeven; NovoNordisk, Copenhagen, Denmark) between June 2002 and June 2003 at The Children's Hospital at Westmead. RESULTS: Six children undergoing cardiac surgery received rFVIIa. Recombinant activated factor VII was administered if bleeding was excessive and persisted despite appropriate investigation and attention to haemostasis by surgical and medical staff. An intravenous dose of 180 microg/kg was given and repeated 2 h later. All of the six patients responded well to rFVIIa with achievement of haemostasis. No adverse events were noted. CONCLUSIONS: Recombinant activated factor VII achieved haemostasis in six paediatric cardiac surgical patients. Good outcomes and no adverse events were noted in these children.

Charge-charge interactions are key determinants of the pK values of ionizable groups in ribonuclease Sa (pI=3.5) and a basic variant (pI=10.2).

The pK values of the titratable groups in ribonuclease Sa (RNase Sa) (pI=3.5), and a charge-reversed variant with five carboxyl to lysine substitutions, 5K RNase Sa (pI=10.2), have been determined by NMR at 20 degrees C in 0.1M NaCl. In RNase Sa, 18 pK values and in 5K, 11 pK values were measured. The carboxyl group of Asp33, which is buried and forms three intramolecular hydrogen bonds in RNase Sa, has the lowest pK (2.4), whereas Asp79, which is also buried but does not form hydrogen bonds, has the most elevated pK (7.4). These results highlight the importance of desolvation and charge-dipole interactions in perturbing pK values of buried groups. Alkaline titration revealed that the terminal amine of RNase Sa and all eight tyrosine residues have significantly increased pK values relative to model compounds.A primary objective in this study was to investigate the influence of charge-charge interactions on the pK values by comparing results from RNase Sa with those from the 5K variant. The solution structures of the two proteins are very similar as revealed by NMR and other spectroscopic data, with only small changes at the N terminus and in the alpha-helix. Consequently, the ionizable groups will have similar environments in the two variants and desolvation and charge-dipole interactions will have comparable effects on the pK values of both. Their pK differences, therefore, are expected to be chiefly due to the different charge-charge interactions. As anticipated from its higher net charge, all measured pK values in 5K RNase are lowered relative to wild-type RNase Sa, with the largest decrease being 2.2 pH units for Glu14. The pK differences (pK(Sa)-pK(5K)) calculated using a simple model based on Coulomb's Law and a dielectric constant of 45 agree well with the experimental values. This demonstrates that the pK differences between wild-type and 5K RNase Sa are mainly due to changes in the electrostatic interactions between the ionizable groups. pK values calculated using Coulomb's Law also showed a good correlation (R=0.83) with experimental values. The more complex model based on a finite-difference solution to the Poisson-Boltzmann equation, which considers desolvation and charge-dipole interactions in addition to charge-charge interactions, was also used to calculate pK values. Surprisingly, these values are more poorly correlated (R=0.65) with the values from experiment. Taken together, the results are evidence that charge-charge interactions are the chief perturbant of the pK values of ionizable groups on the protein surface, which is where the majority of the ionizable groups are positioned in proteins.

pK values of histidine residues in ribonuclease Sa: effect of salt and net charge.

The primary goal of this study was to gain a better understanding of the effect of environment and ionic strength on the pK values of histidine residues in proteins. The salt-dependence of pK values for two histidine residues in ribonuclease Sa (RNase Sa) (pI=3.5) and a variant in which five acidic amino acids have been changed to lysine (5K) (pI=10.2) was measured and compared to pK values of model histidine-containing peptides. The pK of His53 is elevated by two pH units (pK=8.61) in RNase Sa and by nearly one pH unit (pK=7.39) in 5K at low salt relative to the pK of histidine in the model peptides (pK=6.6). The pK for His53 remains elevated in 1.5M NaCl (pK=7.89). The elevated pK for His53 is a result of screenable electrostatic interactions, particularly with Glu74, and a non-screenable hydrogen bond interaction with water. The pK of His85 in RNase Sa and 5K is slightly below the model pK at low salt and merges with this value at 1.5M NaCl. The pK of His85 reflects mainly effects of long-range Coulombic interactions that are screenable by salt. The tautomeric states of the neutral histidine residues are changed by charge reversal. The histidine pK values in RNase Sa are always higher than the pK values in the 5K variant. These results emphasize that the net charge of the protein influences the pK values of the histidine residues. Structure-based pK calculations capture the salt-dependence relatively well but are unable to predict absolute histidine pK values.

Quality of life of survivors of pediatric intensive care.

OBJECTIVE: Measuring outcome in pediatric intensive care is necessary to equate the high cost of treatment with benefits to the patient. Although mortality rates and morbidity are relatively insensitive measures of the benefits of treatment, quality of life measurement gives insight into the long-term outcomes. The aim of this study was to investigate the long-term quality of life outcome of children admitted to a pediatric intensive care unit. DESIGN: Prospective survey. SETTING: A 13-bed pediatric intensive care unit in a university-affiliated, tertiary referral children's hospital. PATIENTS: Patients were 432 children discharged from the pediatric intensive care unit between May 1992 and April 1994. INTERVENTIONS: Quality of life was measured by using the Royal Alexandra Hospital for Children Measure of Function. The scale has two components, the first part completed by the clinician after parent interview and the second part completed separately by the parent. MEASUREMENTS AND MAIN RESULTS: Parents of 432 children were contacted between 3 and 24 months after discharge. Twenty-seven children (6.3%) had died after discharge from the pediatric intensive care unit; 59.3% (256) had scores indicating a normal quality of life, and 32.4% (140) had a fair quality of life with ongoing health, social, or cognitive problems requiring some intervention. Two percent of survivors (nine children) had scores indicating a poor quality of life as they had continued to experience significant or disabling health problems requiring hospitalization or the equivalent. Predictors of poor quality of life included presence of comorbidities, increased length of stay, and a diagnostic category of malignancy. Diagnostic categories of respiratory, trauma, and cardiac dysfunction were associated with a better outcome. CONCLUSIONS: Our results indicate that the long-term outcome in terms of quality of life after admission to a pediatric intensive care unit is good or normal for the majority of surviving children. Those children with a poor outcome are likely to have significant comorbidities or a diagnosis of malignancy.