Failure of national antenatal vitamin D supplementation programme puts dark skinned infants at highest risk: A newborn bloodspot screening study

Objectives: To determine the prevalence of vitamin D deficiency on dried blood spots (DBS) obtained at newborn bloodspot screening (NBS) and thereby test the efficacy of the UK national antenatal supplementation programme in an increasingly ethnically diverse English population. To evaluate the seasonal and ethnic variation in neonatal plasma 25 hydoxyvitamin D (25OHD) and its determinants. Design: Three thousand random DBS samples received at a single regional newborn screening laboratory (52° N) over two one-week periods, one in winter (February 2019) and one in summer (August 2019), were collected. Data was collected from NBS cards on birth weight, gestational age, maternal age, ethnicity, and post code which was replaced with index of multiple deprivation (IMD). 25OHD concentrations were measured on 6mm sub-punch from DBS using quantitative liquid chromatography tandem mass spectrometry adjusted to equivalent plasma values. 25OHD variation with season was assessed using Mann-Whitney U test and ethnic groups compared using Kruskal-Wallis test. Linear regression was used to assess the determinants of 25OHD concentrations. Results: 25OHD measurements were available in 2999 (1580 males) subjects [1499 winter-born and 1500 summer-born]. The majority were white British (59.1%) and born at term (mean SD gestational age of 38.81.8 weeks) with a mean (SD) birth weight of 3306 (565) grams. The overall prevalence of vitamin D deficiency [25OHD<30 nmol/L (12 µg/L)] was 35.7% (n=1070) and insufficiency [30-50 nmol/L (12-20 µg/L)] 33.7% (n=1010). The median (IQR) 25OHD concentration was significantly lower in the winter-born compared to summer-born [29.1 (19.8, 40.6) vs 49.2 (34.3, 64.8) nmol/L respectively; p<0.001]. Across both seasons, when compared to white British babies (41.6 nmol/L), the median 25OHD concentrations were significantly lower in babies of black (30.3 nmol/L; p<0.001), Asian (31.3 nmol/L; p<0.001), any other mixed (32.9 nmol/L; p<0.001), mixed white and black (33.7 nmol/L; p<0.05) and any other white (37.7 nmol/L; p<0.05) ethnicity. The proportion of deficiency was also higher in babies of Asian (48%), black (47%) and mixed ethnicity (38-44%) compared to any other white (34%) or white British (30%) ethnicity. Season of birth, ethnicity, gestation and maternal age accounted for almost 24% of the variation in 25OHD concentrations. Conclusion: The current UK antenatal supplementation programme fails to protect newborns from vitamin D deficiency, especially those from minority ethnic groups who are at high risk of vitamin D deficiency. Nearly 70% of all newborns and 85% of winter-borns had 25OHD concentrations below 50 nmol/L (20 µg/L). Almost 50% of babies of Black or Asian origin were deficient at birth, which explains their high risk of hypocalcaemic complications and rickets if left unsupplemented. Our findings call for an immediate review of the delivery of antenatal and infant vitamin D supplementation programmes and implementation of food fortification in the long term

Improving harmonization and standardization of expanded newborn screening results by optimization of the legacy flow injection analysis tandem mass spectrometry methods and application of a standardized calibration approach

Background Newborn screening (NBS) laboratories in the United Kingdom adhere to common protocols based on single analyte cutoff values (COVs); therefore, interlaboratory harmonization is of paramount importance. Interlaboratory variation for screening analytes in UK NBS laboratories ranges from 17% to 59%. While using common stable isotope internal standards has been shown to significantly reduce interlaboratory variation, instrument set-up, sample extraction, and calibration approach are also key factors. Methods Dried blood spot (DBS) extraction processes, instrument set-up, mobile-phase composition, sample introduction technique, and calibration approach of flow injection analysis–tandem mass spectrometry (FIA-MS/MS) methods were optimized. Inter- and intralaboratory variation of methionine, leucine, phenylalanine, tyrosine, isovaleryl-carnitine, glutaryl-carnitine, octanoyl-carnitine, and decanoyl-carnitine were determined pre- and postoptimization, using 3 different calibration approaches. Results Optimal recovery of analytes from DBS was achieved with a 35-min extraction time and 80% methanol (150 μL). Optimized methodology decreased the mean intralaboratory percentage relative SD (%RSD) for the 8 analytes from 20.7% (range 4.1–46.0) to 5.4% (range 3.0–8.5). The alternative calibration approach reduced the mean interlaboratory %RSD for all analytes from 16.8% (range 4.1–25.0) to 7.1% (range 4.1–11.0). Nuclear magnetic resonance analysis of the calibration material highlighted the need for standardization. The purities of isovaleryl-carnitine and glutaryl-carnitine were 85.13% and 69.94% respectively, below the manufacturer’s stated values of ≥98%. Conclusions For NBS programs provided by multiple laboratories using single analyte COVs, harmonization and standardization of results can be achieved by optimizing legacy FIA-MS/MS methods, adopting a common analytical protocol, and using standardized calibration material rather than internal calibration

N-Hydroxyethyl acrylamide as a functional eROP initiator for the preparation of nanoparticles under “greener” reaction conditions

N-Hydroxyethyl acrylamide was used as a functional initiator for the enzymatic ring-opening polymerisation of ε-caprolactone and δ-valerolactone. N-Hydroxyethyl acrylamide was found not to undergo self-reaction in the presence of Lipase B from Candida antarctica under the reaction conditions employed. By contrast, this is a major problem for 2-hydroxyethyl methacrylate and 2-hydroxyethyl acrylate which both show significant transesterification issues leading to unwanted branching and cross-linking. Surprisingly, N-hydroxyethyl acrylamide did not react fully during enzymatic ring-opening polymerisation. Computational docking studies helped us understand that the initiated polymer chains have a higher affinity for the enzyme active site than the initiator alone, leading to polymer propagation proceeding at a faster rate than polymer initiation leading to incomplete initiator consumption. Hydroxyl end group fidelity was confirmed by organocatalytic chain extension with lactide. N-Hydroxyethyl acrylamide initiated polycaprolactones were free-radical copolymerised with PEGMA to produce a small set of amphiphilic copolymers. The amphiphilic polymers were shown to self-assemble into nanoparticles, and to display low cytotoxicity in 2D in vitro experiments. To increase the green credentials of the synthetic strategies, all reactions were carried out in 2-methyl tetrahydrofuran, a solvent derived from renewable resources and an alternative for the more traditionally used fossil-based solvents tetrahydrofuran, dichloromethane, and toluene

Peri-operative red blood cell transfusion in neonates and infants: NEonate and Children audiT of Anaesthesia pRactice IN Europe: A prospective European multicentre observational study

BACKGROUND: Little is known about current clinical practice concerning peri-operative red blood cell transfusion in neonates and small infants. Guidelines suggest transfusions based on haemoglobin thresholds ranging from 8.5 to 12 g dl-1, distinguishing between children from birth to day 7 (week 1), from day 8 to day 14 (week 2) or from day 15 (≥week 3) onwards. OBJECTIVE: To observe peri-operative red blood cell transfusion practice according to guidelines in relation to patient outcome. DESIGN: A multicentre observational study. SETTING: The NEonate-Children sTudy of Anaesthesia pRactice IN Europe (NECTARINE) trial recruited patients up to 60 weeks' postmenstrual age undergoing anaesthesia for surgical or diagnostic procedures from 165 centres in 31 European countries between March 2016 and January 2017. PATIENTS: The data included 5609 patients undergoing 6542 procedures. Inclusion criteria was a peri-operative red blood cell transfusion. MAIN OUTCOME MEASURES: The primary endpoint was the haemoglobin level triggering a transfusion for neonates in week 1, week 2 and week 3. Secondary endpoints were transfusion volumes, 'delta haemoglobin' (preprocedure - transfusion-triggering) and 30-day and 90-day morbidity and mortality. RESULTS: Peri-operative red blood cell transfusions were recorded during 447 procedures (6.9%). The median haemoglobin levels triggering a transfusion were 9.6 [IQR 8.7 to 10.9] g dl-1 for neonates in week 1, 9.6 [7.7 to 10.4] g dl-1 in week 2 and 8.0 [7.3 to 9.0] g dl-1 in week 3. The median transfusion volume was 17.1 [11.1 to 26.4] ml kg-1 with a median delta haemoglobin of 1.8 [0.0 to 3.6] g dl-1. Thirty-day morbidity was 47.8% with an overall mortality of 11.3%. CONCLUSIONS: Results indicate lower transfusion-triggering haemoglobin thresholds in clinical practice than suggested by current guidelines. The high morbidity and mortality of this NECTARINE sub-cohort calls for investigative action and evidence-based guidelines addressing peri-operative red blood cell transfusions strategies. TRIAL REGISTRATION: ClinicalTrials.gov, identifier: NCT02350348

Manipulation of macrophage programmed cell death pathways by enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli (EPEC) is a human-restricted, clinically significant diarrhoeagenic pathogen that colonises the gut mucosa of hosts via the formation of characteristic attaching and effacing (A/E) lesions. To establish infection and cause disease, EPEC subverts a range of host cell processes through the injection of a suite of bacterial effector proteins into host cells via a Type 3 Secretion System (T3SS). EPEC virulence is critically reliant on the T3SS-delivered Translocated intimin receptor (Tir), which triggers actin polymerisation at the bacterial attachment site in intestinal epithelial cells. However, the manner in which EPEC effector proteins activate and subvert signalling pathways in human immune cells, such as macrophages, remains to be established. During infection the innate immune compartment of the intestinal mucosa plays a pivotal role in antimicrobial immunity, and activation of inflammasome signalling pathways is central to bacterial clearance. This has been established in vivo using the model A/E pathogen Citrobacter rodentium. Interestingly, although evolutionarily closely related to the enteric pathogens Citrobacter, Salmonella and Shigella, the EPEC T3SS needle and flagellin proteins evade detection by inflammasomes. Currently EPEC-induced inflammasome activation in human macrophages is poorly understood. Here I show that infection of human primary macrophages with EPEC rapidly induces NLRP3-inflammasome-driven pyroptosis in a strictly Tir-dependent manner. Mechanistically, Tir-induced actin polymerisation, either via Tir tyrosine phosphorylation and the Nck-N-WASP-ARP2/3 actin polymerisation pathway, or through Tir and the Nck-mimicking effector TccP, stimulated rapid caspase-4-dependent inflammasome responses. Notably, in contrast to caspase-4-driven pyroptosis induced by non-pathogenic E. coli or cytosolic LPS, both pyroptosis and cytokine processing in response to EPEC infection required NLRP3, ASC and caspase-1, and occurred independently of pannexin-1. This study identifies a surprising and indispensable role for both human caspase-4 and NLRP3 in detecting EPEC, and an essential role for pathological actin polymerisation by a bacterial effector in inflammasome activation.Open Acces

Outcome data from 15 years of cystic fibrosis newborn screening in a large UK region

Background: The West Midlands Newborn Bloodspot Screening Laboratory is one of 16 in the UK and serves two tertiary paediatric cystic fibrosis (CF) centres (Staffordshire Children’s Hospital at Royal Stoke and Birmingham Children’s Hospital). CF newborn bloodspot screening (NBS) in this region started in November 2006 prior to the UK national roll-out in 2007. It uses an immunoreactive trypsinogen (IRT)/DNA/IRT protocol. We report the outcomes from 15 years of CF screening. Methods: The West Midlands CF NBS outcomes from 1 November 2006 to 31 October 2021 were reviewed. Clinical data were also obtained for babies referred to the CF centres as ‘CF suspected’. Results: 1 075 161 babies were screened, with 402 referred as ‘CF suspected’ and 205 identified as CF carriers. Of the ‘CF suspected’ babies, 268 were diagnosed with CF, 33 with CF screen positive, inconclusive diagnosis (CFSPID) and 17 as a CF carrier. Any CF-related diagnosis was excluded in 67. Outcome data were not available for 17, of whom 14 had died. Eighteen children with a negative CF NBS have subsequently been diagnosed with CF, 10 had meconium ileus and 8 were true ‘affected not detected’, presenting with respiratory symptoms or failure to thrive. This gives the West Midlands a CF birth prevalence of 1 in 4012 live births and the NBS protocol a sensitivity of 97.1% and a positive predictive value of 66.7%. Conclusions: This large regional data set has excellent case ascertainment and demonstrates successful performance of the CF NBS protocol, with low numbers identified as CFSPID or CF carriers

The Atypical Ubiquitin E2 Conjugase UBE2L3 Is an Indirect Caspase-1 Target and Controls IL-1β Secretion by Inflammasomes

Caspase-1 activation by inflammasome signaling scaffolds initiates inflammation and antimicrobial responses. Caspase-1 proteolytically converts newly induced pro-interleukin 1 beta (IL-1β) into its mature form and directs its secretion, triggering pyroptosis and release of non-substrate alarmins such as interleukin 1 alpha (IL-1α) and HMGB1. While some caspase-1 substrates involved in these events are known, the identities and roles of non-proteolytic targets remain unknown. Here, we use unbiased proteomics to show that the UBE2L3 ubiquitin conjugase is an indirect target of caspase-1. Caspase-1, but not caspase-4, controls pyroptosis- and ubiquitin-independent proteasomal degradation of UBE2L3 upon canonical and non-canonical inflammasome activation by sterile danger signals and bacterial infection. Mechanistically, UBE2L3 acts post-translationally to promote K48-ubiquitylation and turnover of pro-IL-1β and dampen mature-IL-1β production. UBE2L3 depletion increases pro-IL-1β levels and mature-IL-1β secretion by inflammasomes. These findings regarding UBE2L3 as a molecular rheostat have implications for IL-1-driven pathology in hereditary fever syndromes and in autoinflammatory conditions associated with UBE2L3 polymorphisms

Evaluation of a Common Internal Standard Material to Reduce Inter-Laboratory Variation and Ensure the Quality, Safety and Efficacy of Expanded Newborn Screening Results When Using Flow Injection Analysis Tandem Mass Spectrometry with Internal Calibration

In 2015, the newborn screening (NBS) programmes in England and Wales were expanded to include four additional disorders: Classical Homocystinuria, Isovaleric Acidemia, Glutaric Aciduria Type 1 and Maple Syrup Urine Disease, bringing the total number of analytes quantified to eight: phenylalanine, tyrosine, leucine, methionine, isovalerylcarnitine, glutarylcarnitine, octanoylcarnitine and decanoylcarnitine. Post-implementation, population data monitoring showed that inter-laboratory variation was greater than expected, with 90th centiles varying from 17% to 59%. We evaluated the effect of stable isotope internal standard (IS) used for quantitation on inter-laboratory variation. Four laboratories analysed routine screening samples (n &gt; 101,820) using a common IS. Inter-laboratory variation was determined for the eight analytes and compared with results obtained using an in-house common IS (n &gt; 102,194). A linear mixed-effects model was fitted to the data. Using a common IS mix reduced the inter-laboratory variation significantly (p &lt; 0.05) for five analytes. For three analytes, the lack of significance was explained by use of individual laboratory &ldquo;calibration factors&rdquo;. For screening programmes where laboratories adhere to single analyte cut-off values (COVs), it is important that inter-laboratory variation is minimised, primarily to prevent false positive results. Whilst the use of a common IS helps achieve this, it is evident that instrument set-up also contributes to inter-laboratory variation

Enteropathogenic Escherichia coli Stimulates Effector-Driven Rapid Caspase-4 Activation in Human Macrophages

© 2019 The Author(s).Microbial infections can stimulate the assembly of inflammasomes, which activate caspase-1. The gastrointestinal pathogen enteropathogenic Escherichia coli (EPEC) causes localized actin polymerization in host cells. Actin polymerization requires the binding of the bacterial adhesin intimin to Tir, which is delivered to host cells via a type 3 secretion system (T3SS). We show that EPEC induces T3SS-dependent rapid non-canonical NLRP3 inflammasome activation in human macrophages. Notably, caspase-4 activation by EPEC triggers pyroptosis and cytokine processing through the NLRP3-caspase-1 inflammasome. Mechanistically, caspase-4 activation requires the detection of LPS and EPEC-induced actin polymerization, either via Tir tyrosine phosphorylation and the phosphotyrosine-binding adaptor NCK or Tir and the NCK-mimicking effector TccP. An engineered E. coli K12 could reconstitute Tir-intimin signaling, which is necessary and sufficient for inflammasome activation, ruling out the involvement of other virulence factors. Our studies reveal a crosstalk between caspase-4 and caspase-1 that is cooperatively stimulated by LPS and effector-driven actin polymerization.The authors would like to acknowledge grants from the Wellcome Trust (to G.F. and A.R.S.) and the MRC (to P.J.G., G.F., A.R.S., and the CMBI). Work in the laboratory of L.A.F. is funded by grant BIO2017-89081R from the Spanish Ministerio de Ciencia y Universidad (MICIU; AEI/FEDER, EU). G.F. and A.R.S. would like to acknowledge the MRC-funded High-Throughput Single-Cell Analysis facility at the CMBI