Improved multidetector asymmetrical-flow field-flow fractionation method for particle sizing and concentration measurements of lipid-based nanocarriers for RNA delivery.

Lipid-based nanoparticles for RNA delivery (LNP-RNA) are revolutionizing the nanomedicine field, with one approved gene therapy formulation and two approved vaccines against COVID-19, as well as multiple ongoing clinical trials. As for other innovative nanopharmaceuticals (NPhs), the advancement of robust methods to assess their quality and safety profiles-in line with regulatory needs-is critical for facilitating their development and clinical translation. Asymmetric-flow field-flow fractionation coupled to multiple online optical detectors (MD-AF4) is considered a very versatile and robust approach for the physical characterisation of nanocarriers, and has been used successfully for measuring particle size, polydispersity and physical stability of lipid-based systems, including liposomes and solid lipid nanoparticles. However, the unique core structure of LNP-RNA, composed of ionizable lipids electrostatically complexed with RNA, and the relatively labile lipid-monolayer coating, is more prone to destabilization during focusing in MD-AF4 than previously characterised nanoparticles, resulting in particle aggregation and sample loss. Hence characterisation of LNP-RNA by MD-AF4 needs significant adaptation of the methods developed for liposomes. To improve the performance of MD-AF4 applied to LNP-RNA in a systematic and comprehensive manner, we have explored the use of the frit-inlet channel where, differently from the standard AF4 channel, the particles are relaxed hydrodynamically as they are injected. The absence of a focusing step minimizes contact between the particle and the membrane, reducing artefacts (e.g. sample loss, particle aggregation). Separation in a frit-inlet channel enables satisfactory reproducibility and acceptable sample recovery in the commercially available MD-AF4 instruments. In addition to slice-by-slice measurements of particle size, MD-AF4 also allows to determine particle concentration and the particle size distribution, demonstrating enhanced versatility beyond standard sizing measurements.

Pulmonary atresia, "intact ventricular septum", and aortopulmonary collateral arteries.

In muscular pulmonary atresia, major aortopulmonary collateral arteries are characteristic of pulmonary atresia with ventricular septal defect and are rarely seen in pulmonary atresia with intact ventricular septum. Two unusual cases of muscular pulmonary atresia are reported, one with an intact septum and one with a perimembranous ventricular septal defect, closed in utero by aneurysmal tricuspid tissue. In both cases the pulmonary blood supply came entirely from aortopulmonary collaterals. In case 1 a collateral artery connected the left subclavian artery and hypoplastic pulmonary arteries, and several aortopulmonary collaterals arose from the descending aorta, without overlap between these two circulations. In case 2 the pulmonary trunk and arterial duct were absent and the pulmonary blood supply came entirely from collateral arteries. The right ventricle was of normal size and tripartite with a closed perimembranous ventricular septal defect, discovered only at postmortem examination. These observations suggest right ventricular outflow tract obstruction early in fetal development, with involution of the pulmonary trunk and sixth arch derivatives, and persistence of primitive aortopulmonary connections. The morphology in case 1 is at odds with the theoretical division of pulmonary atresia with intact septum and pulmonary atresia with ventricular septal defect into two separate pathological entities that occur at different stages in fetal development.

Severe hypokalaemia and weakness due to Nurofen misuse.

Nephrotoxicity from non-steroidal anti-inflammatory drugs (NSAID) is well recognized. We report a case of severe hypokalaemia and weakness due to renal tubular acidosis in a young woman who was taking 40-60 tablets per day of Nurofen Plus (ibuprofen 200 mg and codeine phosphate 12.8 mg). Proprietary brands of ibuprofen are freely available to the public and those containing codeine may be potentially subject to abuse. This case highlights the need to be aware of this potential and of the life-threatening electrolyte and acid-base disturbances that might be encountered with the widespread availability of these types of NSAID.

The preclinical history of high-frequency ventilation.

As with many innovative techniques in modern medicine, high-frequency ventilation (HFV) was discovered by accident rather than by design. Various modes of HFV also were developed concurrently, with little or no attempt to standardize nomenclature. High-frequency ventilation developed along three main tracks, driven by somewhat different forces.

Muscle weakness in critically ill children.

OBJECTIVE: To establish the incidence of muscle weakness in critically ill children. METHODS: Neuromuscular examinations were performed in 830 children without identified antecedent or acute neuromuscular disease (age 3 months to 17 years 11 months) admitted for >24 hours to a pediatric intensive care unit (ICU) over a 1-year period. RESULTS: Fourteen of 830 (1.7%) patients had generalized weakness. Four failed repeated attempts to extubate. Multiple organ dysfunction occurred in 11 patients and sepsis in 9. Most children received corticosteroids, neuromuscular blocking agents, or aminoglycoside antibiotics. Eight of the 14 children were solid organ or bone marrow transplant recipients. Muscle biopsy showed evidence of acute quadriplegic myopathy in all three patients in whom biopsy was performed. Three patients died. In survivors, significant weakness persisted for 3 to 12 months following ICU discharge. CONCLUSIONS: Muscle weakness is an infrequent but significant feature of critical illness in children. Transplant recipients seem to be at particular risk.

Comparison of classic and endoscopic lymphadenectomy for staging breast cancer.

STUDY OBJECTIVE: To compare endoscopic and classic axillary lymphadenectomy staging of breast cancer with respect to operation-induced changes such as seroma formation, pain, neurologic sensations, lymphedema, infection, and reduction of shoulder-arm mobility. DESIGN: Prospective, randomized study (Canadian Task Force classification I). SETTING: University-affiliated hospital. PATIENTS: Eighty consecutive women with histopathologically confirmed invasive breast cancer who had clinically and sonographically negative axillary lymph nodes (<1 cm). INTERVENTION: Classic and endoscopic lymphadenectomies. MEASUREMENTS AND MAIN RESULTS: We attempted to obtain 10 axillary lymph nodes/patient. After 1, 3, 5, 7, 9, 42, and 84 days, clinical and ultrasonographic examinations were conducted to evaluate operation-induced changes. Short-term results showed that, with endoscopic technique, a representative number of axillary lymph nodes was removed, with reduced axillary infiltration and seroma induction, as well as less impaired shoulder-arm mobility. During the first month, postoperative infection, lymphedema, and neurologic complaints were comparable in both groups, with more stretching pain in the classic group and predominantly paresthesia in the endoscopic group. After 3 months no differences in postoperative complications were detected. CONCLUSIONS: Endoscopic axillary lymphadenectomy avoids short-term reduction of shoulder-arm mobility. Long-term studies are necessary to prove if this technique is as safe as the classic procedure with regard to local axillary recurrence. If so, endoscopy could become the method of choice for staging breast cancer in women with clinically negative lymph nodes.

Repeatability of cellular constituents and cytokine concentration in fluid obtained by non-bronchoscopic bronchoalveolar lavage of infants receiving extracorporeal oxygenation.

BACKGROUND: Since few studies have assessed the repeatability of non-bronchoscopic bronchoalveolar lavage (NB-BAL), we compared cellular counts and cytokine concentrations in fluid obtained by standardised NB-BAL from each side of 20 intubated infants receiving extracorporeal membrane oxygenation (ECMO). METHODS: Total cell counts were obtained from 95 paired lavages and 77 pairs were suitable for differential counts and measurement of cytokine concentrations. RESULTS: Moderate correlation was noted between the two sides for most cell types including total cell counts and percentages of neutrophils and macrophages (R=0.70-0.84) and for cytokine concentrations (IL-8 R=0.78, IL-6 R=0.75, TNF-alpha R=0.64, all p< or =0.001). Using Bland-Altman analysis the mean difference between the two sides approached zero for cellular constituents (total cell counts mean difference 1.7, limits of agreement -187.5 to +190.9 x 10(4)/ml; percentage neutrophils -3.9%, -41.5% to +33.6%; percentage macrophages 3.9%, -33.8% to +41.6%) but tended to be greater on the right for logarithmically transformed cytokine measurements (IL-8: left/right ratio 0.74, limits of agreement 0.12 to 5.45, IL-6: 0.93, 0.09 to 5.87, and TNF-alpha: 0.93, 0.27 to 3.16). Using linear regression with random effects to assess the variability, only the infant's age appeared to influence the cellular results but, for cytokines, only the volume retrieved affected the variability. The magnitude of the measurements, the underlying disease, the operator's experience, days on ECMO, or survival did not affect the variability. CONCLUSION: Measurements obtained by NB-BAL need to be interpreted with caution and strongly suggest that normalisation for the dilutional effects of saline is essential.

The role of neutrophil apoptosis in the resolution of acute lung injury in newborn infants.

BACKGROUND: The persistent airway neutrophilia observed in chronic lung disease of prematurity (CLD) may reflect inappropriate suppression of neutrophil apoptosis. METHODS: 134 bronchoalveolar lavage (BAL) samples were obtained from 32 infants requiring mechanical ventilation for respiratory distress syndrome (RDS): 13 infants (median gestation 26 weeks, range 23 to 28) subsequently developed CLD (CLD group), and 19 infants (gestation 31 weeks, range 25 to 39) recovered fully (RDS group). A further 73 BAL samples were obtained from 20 infants (median age 2 days, range 1 to 402) receiving extracorporeal membrane oxygenation (ECMO) for severe respiratory failure. RESULTS: Neutrophil apoptosis was increased in the RDS group (mean (SEM) neutrophil apoptosis on day 7 BAL: RDS 17.0 (8.6)% v CLD 0.7 (0.2)% (p<0.05)). BAL fluid obtained from RDS but not CLD patients was proapoptotic to neutrophils (apoptosis ratio BAL fluid/saline control: day 1, RDS 9.8 (5.5) v CLD 1.2 (0.1) (p<0.05); day 2, RDS 4.32 (2.8) v CLD 0.5 (0.4) (p<0.05)). There were similar findings in the ECMO group: survivors had proapoptotic BAL fluid compared with non-survivors (apoptosis ratio day 1, survivors 7.9 (2.1) v non-survivors 2.1 (0.7) (p<0.05)). CONCLUSIONS: Inappropriate suppression of neutrophil apoptosis may be associated with a poor outcome in newborn infants with respiratory failure.