Efficacy of a new technique - INtubate-RECruit-SURfactant-Extubate - "IN-REC-SUR-E" - in preterm neonates with respiratory distress syndrome: Study protocol for a randomized controlled trial

Background: Although beneficial in clinical practice, the INtubate-SURfactant-Extubate (IN-SUR-E) method is not successful in all preterm neonates with respiratory distress syndrome, with a reported failure rate ranging from 19 to 69 %. One of the possible mechanisms responsible for the unsuccessful IN-SUR-E method, requiring subsequent re-intubation and mechanical ventilation, is the inability of the preterm lung to achieve and maintain an "optimal" functional residual capacity. The importance of lung recruitment before surfactant administration has been demonstrated in animal studies showing that recruitment leads to a more homogeneous surfactant distribution within the lungs. Therefore, the aim of this study is to compare the application of a recruitment maneuver using the high-frequency oscillatory ventilation (HFOV) modality just before the surfactant administration followed by rapid extubation (INtubate-RECruit-SURfactant-Extubate: IN-REC-SUR-E) with IN-SUR-E alone in spontaneously breathing preterm infants requiring nasal continuous positive airway pressure (nCPAP) as initial respiratory support and reaching pre-defined CPAP failure criteria. Methods/design: In this study, 206 spontaneously breathing infants born at 24+0-27+6 weeks' gestation and failing nCPAP during the first 24 h of life, will be randomized to receive an HFOV recruitment maneuver (IN-REC-SUR-E) or no recruitment maneuver (IN-SUR-E) just prior to surfactant administration followed by prompt extubation. The primary outcome is the need for mechanical ventilation within the first 3 days of life. Infants in both groups will be considered to have reached the primary outcome when they are not extubated within 30 min after surfactant administration or when they meet the nCPAP failure criteria after extubation. Discussion: From all available data no definitive evidence exists about a positive effect of recruitment before surfactant instillation, but a rationale exists for testing the following hypothesis: a lung recruitment maneuver performed with a step-by-step Continuous Distending Pressure increase during High-Frequency Oscillatory Ventilation (and not with a sustained inflation) could have a positive effects in terms of improved surfactant distribution and consequent its major efficacy in preterm newborns with respiratory distress syndrome. This represents our challenge. Trial registration: ClinicalTrials.gov identifier: NCT02482766. Registered on 1 June 2015

Four Arabidopsis berberine-bridge enzyme-like proteins are specific oxidases that inactivate the elicitor-active oligogalacturonides

Recognition of endogenous molecules acting as "damage-associated molecular patterns" (DAMPs) is a key feature of immunity in both animals and plants. Oligogalacturonides (OGs), i.e. fragments derived from the hydrolysis of homogalacturonan, a major component of pectin are a well-known class of DAMPs that activate immunity and protect plants against several microbes. However, hyper-accumulation of OGs severely affects growth, eventually leading to cell death and clearly pointing to OGs as players in the growth-defence trade-off. Here we report a mechanism that may control the homeostasis of OGs avoiding their deleterious hyper-accumulation. By combining affinity chromatography on acrylamide-trapped OGs and other procedures, an Arabidopsis thaliana enzyme that specifically oxidizes OGs was purified and identified. The enzyme was named OG OXIDASE 1 (OGOX1) and shown to be encoded by the gene At4g20830. As a typical flavo-protein, OGOX1 is a sulphite-sensitive H2O2-producing enzyme that displays maximal activity on OGs with a degree of polymerization >4. OGOX1 belongs to a large gene family of mainly apoplastic putative FAD-binding proteins [Berberine-Bridge Enzyme-like (BBE-like); 27 members], whose biochemical and biological function is largely unexplored. We have found that at least four BBE-like enzymes in Arabidopsis are OG oxidases (OGOX1-4). Oxidized OGs display a reduced capability of activating the immune responses and are less hydrolysable by fungal polygalacturonases. Plants overexpressing OGOX1 are more resistant to Botrytis cinerea, pointing to a crucial role of OGOX enzymes in plant immunity

An Arabidopsis berberine bridge enzyme‐like protein specifically oxidizes cellulose oligomers and plays a role in immunity

The plant cell wall is the barrier that pathogens must overcome to cause a disease and to this purpose they secrete degrading enzymes of the various cell wall components. Due to the complexity of these components, several types of oligosaccharide fragments may be released during pathogenesis and some of these can act as Damage-Associated Molecular Pattern (DAMPs). Well-known DAMPs are the oligogalacturonides (OGs) released upon degradation of homogalacturonan and the products of the cellulose breakdown, i.e. the cellodextrins (CDs). We have previously reported that four Arabidopsis Berberine Bridge Enzyme-like (BBE-like) proteins (OGOX1-4) oxidize OGs and impair their elicitor activity. We show here that another Arabidopsis BBE-like protein, which is expressed coordinately with OGOX1 during immunity, specifically oxidizes CDs with a preference for cellotriose (CD3) and longer fragments (CD4-6). Oxidized CDs show a negligible elicitor activity and are less utilizable by the fungus Botrytis cinerea as a carbon source. The enzyme, named CELLOX (CELLODEXTRIN OXIDASE), is encoded by the gene At4g20860. Plants overexpressing CELLOX display an enhanced resistance to B. cinerea likely because oxidized CDs are a less valuable carbon source. Thus, the capacity of oxidizing and impairing the biological activity of the cell wall-derived oligosaccharides seems to be a general trait of the family of BBE-like proteins, which may serve for the homeostatic control of the level of DAMPs to prevent their hyperaccumulation

Improved resistance to pathogens through the induced release of damage-associated molecular patterns

Oligogalacturonides (OGs), oligomers of α-1,4–linked galacturonic acid, are a well-known class of damage-associated molecular patterns (DAMPs) that are recognized by plants as signals of an altered (damaged) self and activate the plant immune response. The accumulation of OGs in vivo is favored by the interaction of microbial polygalacturonases (PGs) with plant-derived inhibitors (PGIPs). Transgenic Arabidopsis plants conditionally expressing a chimeric protein made by a fusion between a PG of Fusarium phyllophilum and a plant-derived PGIP, named “OG machine” (OGM), have been generated, allowing the release on command of OGs in planta and the consequent activation of defense-related responses. A massive and prolonged accumulation of OGs provokes a reduced growth and eventually the senescence of leaves and a cell death resembling the typical hypersensitive response. Expression of the OGM has been obtained in genetic backgrounds defective in key elements involved in immunity. We observed that phenotypes of the OGM plants, i.e. inhibition of seedling root growth and senescence of adult leaves, are recovered, at different extents, in mutants defective in EDS1, a transduction element necessary for salicylic acid accumulation during Effector-Triggered Immunity (ETI), and in RBOHD, a NADPH oxidase involved in the formation of reactive oxygen species (ROS). Root growth is recovered also in plants expressing NahG, a bacterial transgene that leads to reduced level of salicylic acid. Moreover, a cross between DR5::GUS transgenic plants (which express the GUS reporter gene under the control of an IAA inducible promoter) and the OGM was generated, in order to understand whether the OGs can antagonize auxin also when they are released in planta and not only when they are exogenously applied

Oligogalacturonides in immunity and development

The recognition of endogenous molecules acting as “Damage-Associated Molecular Patterns” (DAMPs) is a key feature of the plant immunity. Oligogalacturonides (OGs), cell wall fragments derived from the degradation of the homogalacturonan, are a well-known class of DAMPs. The accumulation of OGs in vivo is favored by the interaction of microbial polygalacturonases (PGs) with plant PGinhibiting proteins (PGIPs). Transgenic Arabidopsis plants expressing an inducible PGIP-PG chimera, named "OG machine" (OGM), accumulate OGs on command in planta. If the release of OGs persists plant growth is inhibited and plants eventually die, reflecting the role of the OGs in the so-called growth-defense “trade-off”. A homeostatic control, which prevents deleterious effects of the over-accumulation of OGs, may rely on the oxidation of OGs, which impairs their signaling activity. Four OG oxidases, named OGOX1-4, that belong to the Berberine Bridge Enzyme-like (BBE-like) super-family have been identified in Arabidopsis. In this work the effects of the altered expression of OGOX1 on the defense responses induced by OGs and on the plant resistance to pathogens have been investigated by reverse genetics. The spatial features of OGOX1 expression during development and in response toelicitors and pathogens using promoter::reporter gene fusions is also described

Nasal high-frequency oscillatory ventilation and CO2 removal: A randomized controlled crossover trial

Objective: To compare short-term application of nasal high-frequency oscillatory ventilation (nHFOV) with nasal continuous positive airway pressure (nCPAP). Working Hypothesis: nHFOV improves CO2 removal with respect to nCPAP in preterm infants needing noninvasive respiratory support and persistent oxygen supply after the first 72 h of life. Study Design: Multicenter non-blinded prospective randomized crossover study. Patient Selection: Thirty premature infants from eight tertiary neonatal intensive care units, of mean \ub1 SD 26.4 \ub1 1.8 weeks of gestational age and 921 \ub1 177 g of birth weight. Methodology: Infants were randomly allocated in a 1:1 ratio to receive a starting treatment mode of either nCPAP or nHFOV delivered by the ventilator CNO (Medin, Germany), using short binasal prongs of appropriate size. A crossover design with four 1-h treatment periods was used, such that each infant received both treatments twice. The primary outcome was the mean transcutaneous partial pressure of CO2 (TcCO2) value during the 2-h cumulative period of nHFOV compared with the 2-h cumulative period of nCPAP. Results: Significantly lower TcCO2 values were observed during nHFOV compared with nCPAP: 47.5 \ub1 7.6 versus 49.9 \ub1 7.2 mmHg, respectively, P = 0.0007. A different TcCO2 behavior was found according to the random sequence: in patients starting on nCPAP, TcCO2 significantly decreased from 50.0 \ub1 8.0 to 46.6 \ub1 7.5 mmHg during nHFOV (P = 0.001). In patients starting on nHFOV, TcCO2 slightly increased from 48.5 \ub1 7.8 to 49.9 \ub1 6.7 mmHg during nCPAP (P = 0.13). Conclusions: nHFOV delivered through nasal prongs is more effective than nCPAP in improving the elimination of CO2

Using the Oxford Cognitive Screen to Detect Cognitive Impairment in Stroke Patients: A Comparison with the Mini-Mental State Examination

BackgroundThe Oxford Cognitive Screen (OCS) was recently developed with the aim of describing the cognitive deficits after stroke. The scale consists of 10 tasks encompassing five cognitive domains: attention and executive function, language, memory, number processing, and praxis. OCS was devised to be inclusive and un-confounded by aphasia and neglect. As such, it may have a greater potential to be informative on stroke cognitive deficits of widely used instruments, such as the Mini-Mental State Examination (MMSE) or the Montreal Cognitive Assessment, which were originally devised for demented patients.ObjectiveThe present study compared the OCS with the MMSE with regards to their ability to detect cognitive impairments post-stroke. We further aimed to examine performance on the OCS as a function of subtypes of cerebral infarction and clinical severity.Methods325 first stroke patients were consecutively enrolled in the study over a 9-month period. The OCS and MMSE, as well as the Bamford classification and NIHSS, were given according to standard procedures.ResultsAbout a third of patients (35.3%) had a performance lower than the cutoff (<22) on the MMSE, whereas 91.6% were impaired in at least one OCS domain, indicating higher incidences of impairment for the OCS. More than 80% of patients showed an impairment in two or more cognitive domains of the OCS. Using the MMSE as a standard of clinical practice, the comparative sensitivity of OCS was 100%. Out of the 208 patients with normal MMSE performance 180 showed impaired performance in at least one domain of the OCS. The discrepancy between OCS and MMSE was particularly strong for patients with milder strokes. As for subtypes of cerebral infarction, fewer patients demonstrated widespread impairments in the OCS in the Posterior Circulation Infarcts category than in the other categories.ConclusionOverall, the results showed a much higher incidence of cognitive impairment with the OCS than with the MMSE and demonstrated no false negatives for OCS vs MMSE. It is concluded that OCS is a sensitive screen tool for cognitive deficits after stroke. In particular, the OCS detects high incidences of stroke-specific cognitive impairments, not detected by the MMSE, demonstrating the importance of cognitive profiling

Lung recruitment before surfactant administration in extremely preterm neonates with respiratory distress syndrome (IN-REC-SUR-E): a randomised, unblinded, controlled trial

Background: The importance of lung recruitment before surfactant administration has been shown in animal studies. Well designed trials in preterm infants are absent. We aimed to examine whether the application of a recruitment manoeuvre just before surfactant administration, followed by rapid extubation (intubate-recruit-surfactant-extubate [IN-REC-SUR-E]), decreased the need for mechanical ventilation during the first 72 h of life compared with no recruitment manoeuvre (ie, intubate-surfactant-extubate [IN-SUR-E]). Methods: We did a randomised, unblinded, controlled trial in 35 tertiary neonatal intensive care units in Italy. Spontaneously breathing extremely preterm neonates (24 + 0 to 27 + 6 weeks' gestation) reaching failure criteria for continuous positive airway pressure within the first 24 h of life were randomly assigned (1:1) with a minimisation algorithm to IN-REC-SUR-E or IN-SUR-E using an interactive web-based electronic system, stratified by clinical site and gestational age. The primary outcome was the need for mechanical ventilation in the first 72 h of life. Analyses were done in intention-to-treat and per-protocol populations, with a log-binomial regression model correcting for stratification factors to estimate adjusted relative risk (RR). This study is registered with ClinicalTrials.gov, NCT02482766. Findings: Of 556 infants assessed for eligibility, 218 infants were recruited from Nov 12, 2015, to Sept 23, 2018, and included in the intention-to-treat analysis. The requirement for mechanical ventilation during the first 72 h of life was reduced in the IN-REC-SUR-E group (43 [40%] of 107) compared with the IN-SUR-E group (60 [54%] of 111; adjusted RR 0·75, 95% CI 0·57–0·98; p=0·037), with a number needed to treat of 7·2 (95% CI 3·7–135·0). The addition of the recruitment manoeuvre did not adversely affect the safety outcomes of in-hospital mortality (19 [19%] of 101 in the IN-REC-SUR-E group vs 37 [33%] of 111 in the IN-SUR-E group), pneumothorax (four [4%] of 101 vs seven [6%] of 111), or grade 3 or worse intraventricular haemorrhage (12 [12%] of 101 vs 17 [15%] of 111). Interpretation: A lung recruitment manoeuvre just before surfactant administration improved the efficacy of surfactant treatment in extremely preterm neonates compared with the standard IN-SUR-E technique, without increasing the risk of adverse neonatal outcomes. The reduced need for mechanical ventilation during the first 72 h of life might facilitate implementation of a non-invasive respiratory support strategy. Funding: None