Aerosol Delivery of Palivizumab in a Neonatal Lamb Model of Respiratory Syncytial Virus Infection.

(1) Background: Palivizumab has been an approved preventative monoclonal antibody for respiratory syncytial virus (RSV) infection for over two decades. However, due to its high cost and requirement for multiple intramuscular injections, its use has been limited mostly to high-income countries. Following our previous study showing the successful lung deposition of aerosolised palivizumab in lambs, this current study evaluated the "proof-of-principle" effect of aerosolised palivizumab delivered as a therapeutic to neonatal lambs following RSV infection. (2) Methods: Neonatal lambs were intranasally inoculated with RSV-A2 on day 0 (day 3 post-birth) and treated with aerosolised palivizumab 3 days later (day 3 post-inoculation). Clinical symptoms, RSV viral load and inflammatory response were measured post-inoculation. (3) Results: Aerosolised therapeutic delivery of palivizumab did not reduce RSV viral loads in the nasopharynx nor the bronchoalveolar lavage fluid, but resulted in a modest reduction in inflammatory response at day 6 post-inoculation compared with untreated lambs. (4) Conclusions: This proof-of-principle study shows some evidence of aerosolised palivizumab reducing RSV inflammation, but further studies using optimized protocols are needed in order to validate these findings.

Current and emerging technologies for the timely screening and diagnosis of neonatal jaundice.

Neonatal jaundice is one of the most common clinical conditions affecting newborns. For most newborns, jaundice is harmless, however, a proportion of newborns develops severe neonatal jaundice requiring therapeutic interventions, accentuating the need to have reliable and accurate screening tools for timely recognition across different health settings. The gold standard method in diagnosing jaundice involves a blood test and requires specialized hospital-based laboratory instruments. Despite technological advancements in point-of-care laboratory medicine, there is limited accessibility of the specialized devices and sample stability in geographically remote areas. Lack of suitable testing options leads to delays in timely diagnosis and treatment of clinically significant jaundice in developed and developing countries alike. There has been an ever-increasing need for a low-cost, simple to use screening technology to improve timely diagnosis and management of neonatal jaundice. Consequently, several point-of-care (POC) devices have been developed to address this concern. This paper aims to review the literature, focusing on emerging technologies in the screening and diagnosing of neonatal jaundice. We report on the challenges associated with the existing screening tools, followed by an overview of emerging sensors currently in pre-clinical development and the emerging POC devices in clinical trials to advance the screening of neonatal jaundice. The benefits offered by emerging POC devices include their ease of use, low cost, and the accessibility of rapid response test results. However, further clinical trials are required to overcome the current limitations of the emerging POC's before their implementation in clinical settings. Hence, the need for a simple to use, low-cost POC jaundice detection technology for newborns remains an unsolved challenge globally.

A Novel Optical Assay System for Bilirubin Concentration Measurement in Whole Blood.

As a biomarker for liver disease, bilirubin has been utilized in prognostic scoring systems for cirrhosis. While laboratory-based methods are used to determine bilirubin levels in clinical settings, they do not readily lend themselves to applications outside of hospitals. Consequently, bilirubin monitoring for cirrhotic patients is often performed only intermittently; thus, episodes requiring clinical interventions could be missed. This work investigates the feasibility of measuring bilirubin concentration in whole porcine blood samples using dual-wavelength transmission measurement. A compact and low-cost dual-wavelength transmission measurement setup is developed and optimized to measure whole blood bilirubin concentrations. Using small volumes of whole porcine blood (72 µL), we measured the bilirubin concentration within a range corresponding to healthy individuals and cirrhotic patients (1.2-30 mg/dL). We demonstrate that bilirubin levels can be estimated with a positive correlation (R-square > 0.95) and an accuracy of ±1.7 mg/dL, with higher reliability in cirrhotic bilirubin concentrations (> 4 mg/dL) - critical for high-risk patients. The optical and electronic components utilized are economical and can be readily integrated into a miniature, low-cost, and user-friendly system. This could provide a pathway for point-of-care monitoring of blood bilirubin outside of medical facilities (e.g., patient's home).

Detection of Vitamin D Metabolites in Breast Milk: Perspectives and challenges for measurement by Liquid Chromatography Tandem-Mass Spectrometry.

Breast milk is an emerging matrix for vitamin D assessment of breastfed infants and their mothers. It is considered a more reliable indicator of infant intake than the assessment of maternal circulating vitamin D. With the improved sensitivity of mass spectrometry-based technologies, this method principle has been the recent mainstay for the quantitation of various vitamin D metabolites in breast milk for population-based clinical trials. There are still several areas across the total testing process (pre-analytical, analytical and post-analytical) to be defined and harmonised to translate breast milk vitamin D measurement by liquid chromatography-tandem mass spectrometry (LC-MS/MS) from population-based research to routine clinical use and public health applications. Pre-analytically, the determination of the best form of vitamin D to measure in breast milk requires more evidence. Analytically, standardisation of the methods to allow for comparability of results is required. Post analytically, breast milk vitamin D decision limits are needed to turn the individual numerical outputs into clinically meaningful results. This review aims to synthesise the current evidence and utility of measurement of breast milk vitamin D by LC-MS/MS and to lead a future discussion on best practices to allow for its clinical utility beyond its current research-based use.

Practical microcircuits for handheld acoustofluidics.

Acoustofluidics has promised to enable lab-on-a-chip and point-of-care devices in ways difficult to achieve using other methods. Piezoelectric ultrasonic transducers-as small as the chips they actuate-provide rapid fluid and suspended object transport. Acoustofluidic lab-on-chip devices offer a vast range of benefits in early disease identification and noninvasive drug delivery. However, their potential has long been undermined by the need for benchtop or rack-mount electronics. The piezoelectric ultrasonic transducers within require these equipment and thus acoustofluidic device implementation in a bedside setting has been limited. Here we detail a general process to enable the reader to produce battery or mains-powered microcircuits ideal for driving 1-300 MHz acoustic devices. We include the general design strategy for the circuit, the blocks that collectively define it, and suitable, specific choices for components to produce these blocks. We furthermore illustrate how to incorporate automated resonance finding and tracking, sensing and feedback, and built-in adjustability to accommodate devices' vastly different operating frequencies and powers in a single driver, including examples of fluid and particle manipulation typical of the needs in our discipline. With this in hand, the many groups active in lab-on-a-chip acoustofluidics can now finally deliver on the promise of handheld, point-of-care technologies.

Mechanism of bilirubin elimination in urine: insights and prospects for neonatal jaundice.

Despite a century of research, bilirubin metabolism and the transport mechanisms responsible for homeostasis of bilirubin in serum remain controversial. Emerging evidence on the hepatic membrane transporters and inherited disorders of bilirubin metabolism have contributed to a greater understanding of the various steps involved in bilirubin homeostasis and its associated excretory pathways. We discuss these recent research findings on hepatic membrane transporters and evaluate their significance on the newborn bilirubin metabolism and excretion. New insights gained speculate that a proportion of conjugated bilirubin is excreted via the renal system, as an alternative to the intestinal excretion, even in normal physiological jaundice with no associated pathological concerns. Finally, this paper discusses the clinical relevance of targeting the altered renal excretory pathway, as bilirubin in urine may hold diagnostic importance in screening for neonatal jaundice.

Pulmonary Deposition of Radionucleotide-Labeled Palivizumab: Proof-of-Concept Study.

OBJECTIVE: Current prevention and/or treatment options for respiratory syncytial virus (RSV) infections are limited as no vaccine is available. Prophylaxis with palivizumab is very expensive and requires multiple intramuscular injections over the RSV season. Here we present proof-of-concept data using nebulized palivizumab delivery as a promising new approach for the prevention or treatment of severe RSV infections, documenting both aerosol characteristics and pulmonary deposition patterns in the lungs of lambs. DESIGN: Prospective animal study. SETTING: Biosecurity Control Level 2-designated large animal research facility at the Murdoch Children's Research Institute, Melbourne, Australia. SUBJECTS: Four weaned Border-Leicester/Suffolk lambs at 5 months of age. INTERVENTIONS: Four lambs were administered aerosolized palivizumab conjugated to Tc-99m, under gaseous anesthesia, using either the commercially available AeroNeb Go® or the investigational HYDRA device, placed in-line with the inspiratory limb of a breathing circuit. Lambs were scanned in a single-photon emission computed tomography (SPECT/CT) scanner in the supine position during the administration procedure. MEASUREMENTS AND MAIN RESULTS: Both the HYDRA and AeroNeb Go® produced palivizumab aerosols in the 1-5 µm range with similar median (geometric standard deviation and range) aerosol droplet diameters for the HYDRA device (1.84 ± 1.40 µm, range = 0.54-5.41µm) and the AeroNeb Go® (3.07 ± 1.56 µm, range = 0.86-10 µm). Aerosolized palivizumab was delivered to the lungs at 88.79-94.13% of the total aerosolized amount for all lambs, with a small proportion localized to either the trachea or stomach. No difference between devices were found. Pulmonary deposition ranged from 6.57 to 9.25% of the total dose of palivizumab loaded in the devices, mostly in the central right lung. CONCLUSIONS: Aerosolized palivizumab deposition patterns were similar in all lambs, suggesting a promising approach in the control of severe RSV lung infections.

Surgery and magnetic resonance imaging increase the risk of hypothermia in infants.

AIM: Maintaining normothermia is a tenet of neonatal care. However, neonatal thermal care guidelines applicable to intra-hospital transport beyond the neonatal intensive care unit (NICU) and during surgery or magnetic resonance imaging (MRI) are lacking. The aim of this study is to determine the proportion of infants normothermic (36.5-37.5°C) on return to NICU after management during surgery and MRI, and during standard clinical care in both environments. METHODS: Sixty-two newborns requiring either surgery in the operating theatre (OT) (n = 41) or an MRI scan (n = 21) at the Royal Children's Hospital (Melbourne) NICU were prospectively studied. Core temperature, along with cardiorespiratory parameters, was continuously measured from 15 min prior to leaving the NICU until 60 min after returning. Passive and active warming (intra-operatively) was at clinician discretion. RESULTS: The study reported 90% of infants were normothermic before leaving NICU: 86% (MRI) and 93% (OT). Only 52% of infants were normothermic on return to NICU (relative risk (RR) 1.75; 95% confidence interval (CI) 1.39-2.31; number needed to harm (NNH) 2.6). Between departure from the NICU and commencement of surgery, core temperature decreased by mean 0.81°C (95% CI 0.30-1.33; P = 0.0001, analysis of variance), with only 24% of infants normothermic when surgery began (P < 0.0001; RR 3.80 (95% CI 2.33-6.74); NNH 1.5). After an MRI, infants were a mean 0.41°C (95% CI 0.16-0.67) colder than immediately before entering the scanner (P = 0.001, analysis of variance), with only 43% being normothermic (P = 0.003; RR 2.11 (95% CI 1.35-3.74); NNH 2.1). CONCLUSION: Unintentional hypothermia is a common occurrence during surgery in the OT and MRI in neonates, indicating that evidence-based warming strategies to prevent hypothermia should be developed.

Lung Recruitment Strategies During High Frequency Oscillatory Ventilation in Preterm Lambs.

Background: High frequency oscillatory ventilation (HFOV) is considered a lung protective ventilation mode in preterm infants only if lung volume is optimized. However, whilst a "high lung volume strategy" is advocated for HFOV in preterm infants this strategy is not precisely defined. It is not known to what extent lung recruitment should be pursued to provide lung protection. In this study we aimed to determine the relationship between the magnitude of lung volume optimization and its effect on gas exchange and lung injury in preterm lambs. Methods: 36 surfactant-deficient 124-127 d lambs commenced HFOV immediately following a sustained inflation at birth and were allocated to either (1) no recruitment (low lung volume; LLV), (2) medium- (MLV), or (3) high lung volume (HLV) recruitment strategy. Gas exchange and lung volume changes over time were measured. Lung injury was analyzed by post mortem pressure-volume curves, alveolar protein leakage, gene expression, and histological injury score. Results: More animals in the LLV developed a pneumothorax compared to both recruitment groups. Gas exchange was superior in both recruitment groups compared to LLV. Total lung capacity tended to be lower in the LLV group. Other parameters of lung injury were not different. Conclusions: Lung recruitment during HFOV optimizes gas exchange but has only modest effects on lung injury in a preterm animal model. In the HLV group aiming at a more extensive lung recruitment gas exchange was better without affecting lung injury.

Time to lung aeration during a sustained inflation at birth is influenced by gestation in lambs.

BackgroundCurrent sustained lung inflation (SI) approaches use uniform pressures and durations. We hypothesized that gestational-age-related mechanical and developmental differences would affect the time required to achieve optimal lung aeration, and resultant lung volumes, during SI delivery at birth in lambs.Methods49 lambs, in five cohorts between 118 and 139 days of gestation (term 142 d), received a standardized 40 cmH2O SI, which was delivered until 10 s after lung volume stability (optimal aeration) was visualized on real-time electrical impedance tomography (EIT), or to a maximum duration of 180 s. Time to stable lung aeration (Tstable) within the whole lung, gravity-dependent, and non-gravity-dependent regions, was determined from EIT recordings.ResultsTstable was inversely related to gestation (P<0.0001, Kruskal-Wallis test), with the median (range) being 229 (85,306) s and 72 (50,162) s in the 118-d and 139-d cohorts, respectively. Lung volume at Tstable increased with gestation from a mean (SD) of 20 (17) ml/kg at 118 d to 56 (13) ml/kg at 139 d (P=0.002, one-way ANOVA). There were no gravity-dependent regional differences in Tstable or aeration.ConclusionsThe trajectory of aeration during an SI at birth is influenced by gestational age in lambs. An understanding of this may assist in developing SI protocols that optimize lung aeration for all infants.

Selection of Reference Genes for Gene Expression Studies related to lung injury in a preterm lamb model.

Preterm newborns often require invasive support, however even brief periods of supported ventilation applied inappropriately to the lung can cause injury. Real-time quantitative reverse transcriptase-PCR (qPCR) has been extensively employed in studies of ventilation-induced lung injury with the reference gene 18S ribosomal RNA (18S RNA) most commonly employed as the internal control reference gene. Whilst the results of these studies depend on the stability of the reference gene employed, the use of 18S RNA has not been validated. In this study the expression profile of five candidate reference genes (18S RNA, ACTB, GAPDH, TOP1 and RPS29) in two geographical locations, was evaluated by dedicated algorithms, including geNorm, Normfinder, Bestkeeper and ΔCt method and the overall stability of these candidate genes determined (RefFinder). Secondary studies examined the influence of reference gene choice on the relative expression of two well-validated lung injury markers; EGR1 and IL1B. In the setting of the preterm lamb model of lung injury, RPS29 reference gene expression was influenced by tissue location; however we determined that individual ventilation strategies influence reference gene stability. Whilst 18S RNA is the most commonly employed reference gene in preterm lamb lung studies, our results suggest that GAPDH is a more suitable candidate.

An authentic animal model of the very preterm infant on nasal continuous positive airway pressure.

BACKGROUND: The surge in uptake of nasal continuous positive airway pressure (CPAP) for respiratory support in preterm infants has occurred in the absence of an authentic animal model. Such a model would allow investigation of research questions of physiological and therapeutic importance. We therefore aimed to develop a preterm lamb model of the non-intubated very preterm infant on CPAP. METHODS: After staged exteriorisation and instrumentation, preterm lambs were delivered from anaesthetised ewes at 131 to 133 days gestation. Via a single nasal prong (4-mm internal diameter, 6- to 7-cm depth), positive pressure was delivered from the outset, with nasal intermittent positive pressure ventilation (NIPPV) used until transition to nasal CPAP was attempted, and periodically thereafter for hypoventilation. Caffeine and doxapram were used as respiratory stimulants. Gastric distension was prevented with an oesophageal balloon. Cardiorespiratory parameters and results of arterial blood gas analyses were monitored throughout the study period, which continued for 150 min after first transition to CPAP. RESULTS: Ten preterm lambs were studied, at gestation 132 ± 1 days (mean ± SD) and birth weight 3.6 ± 0.45 kg. After stabilisation on NIPPV, transition to nasal CPAP was first attempted at 28 ± 11 min. There was transient respiratory acidosis, with gradual resolution as spontaneous respiratory activity increased. In the final hour, 79% ± 33% of time was spent on CPAP alone, with typical respiratory rates around 60 breaths per minute. PaCO2 at end-experiment was 58 ± 36 mmHg. CONCLUSIONS: Non-intubated preterm lambs can be effectively transitioned to nasal CPAP soon after birth. This animal model will be valuable for further research.

Effective pulmonary delivery of an aerosolized plasmid DNA vaccine via surface acoustic wave nebulization.

BACKGROUND: Pulmonary-delivered gene therapy promises to mitigate vaccine safety issues and reduce the need for needles and skilled personnel to use them. While plasmid DNA (pDNA) offers a rapid route to vaccine production without side effects or reliance on cold chain storage, its delivery to the lung has proved challenging. Conventional methods, including jet and ultrasonic nebulizers, fail to deliver large biomolecules like pDNA intact due to the shear and cavitational stresses present during nebulization. METHODS: In vitro structural analysis followed by in vivo protein expression studies served in assessing the integrity of the pDNA subjected to surface acoustic wave (SAW) nebulisation. In vivo immunization trials were then carried out in rats using SAW nebulized pDNA (influenza A, human hemagglutinin H1N1) condensate delivered via intratracheal instillation. Finally, in vivo pulmonary vaccinations using pDNA for influenza was nebulized and delivered via a respirator to sheep. RESULTS: The SAW nebulizer was effective at generating pDNA aerosols with sizes optimal for deep lung delivery. Successful gene expression was observed in mouse lung epithelial cells, when SAW-nebulized pDNA was delivered to male Swiss mice via intratracheal instillation. Effective systemic and mucosal antibody responses were found in rats via post-nebulized, condensed fluid instillation. Significantly, we demonstrated the suitability of the SAW nebulizer to administer unprotected pDNA encoding an influenza A virus surface glycoprotein to respirated sheep via aerosolized inhalation. CONCLUSION: Given the difficulty of inducing functional antibody responses for DNA vaccination in large animals, we report here the first instance of successful aerosolized inhalation delivery of a pDNA vaccine in a large animal model relevant to human lung development, structure, physiology, and disease, using a novel, low-power (<1 W) surface acoustic wave (SAW) hand-held nebulizer to produce droplets of pDNA with a size range suitable for delivery to the lower respiratory airways.