Composition of early life leukocyte populations in preterm infants with and without late-onset sepsis

Background Composition of leukocyte populations in the first month of life remains incompletely characterised, particularly in preterm infants who go on to develop late-onset sepsis (LOS). Aim To characterise and compare leukocyte populations in preterm infants with and without LOS during the first month of life. Study design Single-centre prospective observational cohort study. Participants Infants born <30 weeks gestational age (GA). Outcome measures Peripheral blood samples were collected at 1, 7, 14, 21 and 28 days of life. Leukocyte populations were characterised using 5-fluorophore-6-marker flow cytometry. Absolute leukocyte counts and frequency of total CD45+ leukocytes of each population were adjusted for GA, birth weight z-scores, sex and total leukocyte count. Results Of 119 preterm infants enrolled, 43 (36%) had confirmed or clinical LOS, with a median onset at 13 days (range 6–26). Compared to infants without LOS, the adjusted counts and frequency of neutrophils, basophils and non-cytotoxic T lymphocytes were generally lower and immature granulocytes were higher over the first month of life in infants who developed LOS. Specific time point comparisons identified lower adjusted neutrophil counts on the first day of life in those infants who developed LOS more than a week later, compared to those without LOS, albeit levels were within the normal age-adjusted range. Non-cytotoxic T lymphocyte counts and/or frequencies were lower in infants following LOS on days 21 and 28 when compared to those who did not develop LOS. Conclusion Changes in non-cytotoxic T lymphocytes occurred following LOS suggesting sepsis-induced immune suppression

Meeting report: a hard look at the state of enamel research.

The Encouraging Novel Amelogenesis Models and Ex vivo cell Lines (ENAMEL) Development workshop was held on 23 June 2017 at the Bethesda headquarters of the National Institute of Dental and Craniofacial Research (NIDCR). Discussion topics included model organisms, stem cells/cell lines, and tissues/3D cell culture/organoids. Scientists from a number of disciplines, representing institutions from across the United States, gathered to discuss advances in our understanding of enamel, as well as future directions for the field

Enamel ribbons, surface nodules, and octacalcium phosphate in C57BL/6 Amelxâ /â mice and Amelx+/â lyonization

BackgroundAmelogenin is required for normal enamel formation and is the most abundant protein in developing enamel.MethodsAmelx+/+, Amelx+/â , and Amelxâ /â molars and incisors from C57BL/6 mice were characterized using RTâ PCR, Western blotting, dissecting and light microscopy, immunohistochemistry (IHC), transmission electron microscopy (TEM), scanning electron microscopy (SEM), backscattered SEM (bSEM), nanohardness testing, and Xâ ray diffraction.ResultsNo amelogenin protein was detected by Western blot analyses of enamel extracts from Amelxâ /â mice. Amelxâ /â incisor enamel averaged 20.3 ± 3.3 μm in thickness, or only 1/6th that of the wild type (122.3 ± 7.9 μm). Amelxâ /â incisor enamel nanohardness was 1.6 Gpa, less than half that of wildâ type enamel (3.6 Gpa). Amelx+/â incisors and molars showed vertical banding patterns unique to each tooth. IHC detected no amelogenin in Amelxâ /â enamel and varied levels of amelogenin in Amelx+/â incisors, which correlated positively with enamel thickness, strongly supporting lyonization as the cause of the variations in enamel thickness. TEM analyses showed characteristic mineral ribbons in Amelx+/+ and Amelxâ /â enamel extending from mineralized dentin collagen to the ameloblast. The Amelxâ /â enamel ribbons were not well separated by matrix and appeared to fuse together, forming plates. Xâ ray diffraction determined that the predominant mineral in Amelxâ /â enamel is octacalcium phosphate (not calcium hydroxyapatite). Amelxâ /â ameloblasts were similar to wildâ type ameloblasts except no Tomesâ processes extended into the thin enamel. Amelxâ /â and Amelx+/â molars both showed calcified nodules on their occlusal surfaces. Histology of D5 and D11 developing molars showed nodules forming during the maturation stage.ConclusionAmelogenin forms a resorbable matrix that separates and supports, but does not shape early secretoryâ stage enamel ribbons. Amelogenin may facilitate the conversion of enamel ribbons into hydroxyapatite by inhibiting the formation of octacalcium phosphate. Amelogenin is necessary for thickening the enamel layer, which helps maintain ribbon organization and development and maintenance of the Tomesâ process.We thoroughly characterized enamel formation in amelogenin null mice and determined that the mineral covering dentin in these animals is octacalcium phosphate. The initial enamel mineral has a ribbon shape, similar to the wild type. Thus, amelogenin is not required to shape the ribbons, as is currently thought, but is required to ensure that the final mineral phase is calcium hydroxyapatite.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/134766/1/mgg3252_am.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/134766/2/mgg3252-sup-0001-AppendixS1-21.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/134766/3/mgg3252.pd

Biosynthesis and enzymology of the Caenorhabditis elegans cuticle: identification and characterization of a novel serine protease inhibitor.

The nematode Caenorhabditis elegans represents an excellent model in which to examine nematode gene expression and function. A completed genome, straightforward transgenesis, available mutants and practical genome-wide RNAi approaches provide an invaluable toolkit in the characterization of nematode genes. We have performed a targeted RNAi screen in an attempt to identify components of the cuticle collagen biosynthetic pathway. Collagen biosynthesis and cuticle assembly are multi-step processes that involve numerous key enzymes involved in post-translational modification, trimer folding, procollagen processing and subsequent cross-linking stages. Many of these steps, the modifications and the enzymes are unique to nematodes and may represent attractive targets for the control of parasitic nematodes. A novel serine protease inhibitor was uncovered during our targeted screen, which is involved in collagen maturation, proper cuticle assembly and the moulting process. We have confirmed a link between this inhibitor and the previously uncharacterized bli-5 locus in C. elegans. The mutant phenotype, spatial expression pattern and the over-expression phenotype of the BLI-5 protease inhibitor and their relevance to collagen biosynthesis are discussed

Enamelin is critical for ameloblast integrity and enamel ultrastructure formation

Mutations in the human enamelin gene cause autosomal dominant hypoplastic amelogenesis imperfecta in which the affected enamel is thin or absent. Study of enamelin knockout NLS-lacZ knockin mice revealed that mineralization along the distal membrane of ameloblast is deficient, resulting in no true enamel formation. To determine the function of enamelin during enamel formation, we characterized the developing teeth of the Enam-/- mice, generated amelogenin-driven enamelin transgenic mouse models, and then introduced enamelin transgenes into the Enam-/- mice to rescue enamel defects. Mice at specific stages of development were subjected to morphologic and structural analysis using β-galactosidase staining, immunohistochemistry, and transmission and scanning electron microscopy. Enamelin expression was ameloblast-specific. In the absence of enamelin, ameloblasts pathology became evident at the onset of the secretory stage. Although the aggregated ameloblasts generated matrix-containing amelogenin, they were not able to create a well-defined enamel space or produce normal enamel crystals. When enamelin is present at half of the normal quantity, enamel was thinner with enamel rods not as tightly arranged as in wild type suggesting that a specific quantity of enamelin is critical for normal enamel formation. Enamelin dosage effect was further demonstrated in transgenic mouse lines over expressing enamelin. Introducing enamelin transgene at various expression levels into the Enam -/- background did not fully recover enamel formation while a medium expresser in the Enam+/- background did. Too much or too little enamelin abolishes the production of enamel crystals and prism structure. Enamelin is essential for ameloblast integrity and enamel formation. © 2014 Hu et al

Neurodevelopmental outcomes at 7 years’ corrected age in preterm infants who were fed high-dose docosahexaenoic acid to term equivalent: a follow-up of a randomised controlled trial

This is an Open Access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work noncommercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http:// creativecommons.org/licenses/by-nc/4.0/OBJECTIVE: To determine if improvements in cognitive outcome detected at 18 months' corrected age (CA) in infants born <33 weeks' gestation receiving a high-docosahexaenoic acid (DHA) compared with standard-DHA diet were sustained in early childhood. DESIGN: Follow-up of a multicentre randomised controlled trial. Randomisation was stratified for sex, birth weight (<1250 vs ≥1250 g) and hospital. SETTING: Five Australian tertiary hospitals from 2008 to 2013. PARTICIPANTS: 626 of the 657 participants randomised between 2001 and 2005 were eligible to participate. INTERVENTIONS: High-DHA (≈1% total fatty acids) enteral feeds compared with standard-DHA (≈0.3% total fatty acids) from age 2-4 days until term CA. PRIMARY OUTCOME: Full Scale IQ of the Wechsler Abbreviated Scale of Intelligence (WASI) at 7 years CA. Prespecified subgroup analyses based on the randomisation strata (sex, birth weight) were conducted. RESULTS: 604 (92% of the 657 originally randomised) consented to participate (291 high-DHA, 313 standard-DHA). To address missing data in the 604 consenting participants (22 for primary outcome), multiple imputation was performed. The Full Scale IQ was not significantly different between groups (high-DHA 98.3, SD 14.0, standard-DHA 98.5, SD 14.9; mean difference adjusted for sex, birthweight strata and hospital -0.3, 95% CI -2.9 to 2.2; p=0.79). There were no significant differences in any secondary outcomes. In prespecified subgroup analyses, there was a significant sex by treatment interaction on measures of parent-reported executive function and behaviour. Scores were within the normal range but girls receiving the high-DHA diet scored significantly higher (poorer outcome) compared with girls receiving the standard-DHA diet. CONCLUSIONS: Supplementing the diets of preterm infants with a DHA dose of approximately 1% total fatty acids from days 2-4 until term CA showed no evidence of benefit at 7 years' CA. TRIAL REGISTRATION NUMBER: Australian New Zealand Clinical Trials Registry: ACTRN12606000327583

Nicotinic Receptor Alpha7 Expression during Tooth Morphogenesis Reveals Functional Pleiotropy

The expression of nicotinic acetylcholine receptor (nAChR) subtype, alpha7, was investigated in the developing teeth of mice that were modified through homologous recombination to express a bi-cistronic IRES-driven tau-enhanced green fluorescent protein (GFP); alpha7GFP) or IRES-Cre (alpha7Cre). The expression of alpha7GFP was detected first in cells of the condensing mesenchyme at embryonic (E) day E13.5 where it intensifies through E14.5. This expression ends abruptly at E15.5, but was again observed in ameloblasts of incisors at E16.5 or molar ameloblasts by E17.5–E18.5. This expression remains detectable until molar enamel deposition is completed or throughout life as in the constantly erupting mouse incisors. The expression of alpha7GFP also identifies all stages of innervation of the tooth organ. Ablation of the alpha7-cell lineage using a conditional alpha7Cre×ROSA26-LoxP(diphtheria toxin A) strategy substantially reduced the mesenchyme and this corresponded with excessive epithelium overgrowth consistent with an instructive role by these cells during ectoderm patterning. However, alpha7knock-out (KO) mice exhibited normal tooth size and shape indicating that under normal conditions alpha7 expression is dispensable to this process. The function of ameloblasts in alpha7KO mice is altered relative to controls. High resolution micro-computed tomography analysis of adult mandibular incisors revealed enamel volume of the alpha7KO was significantly reduced and the organization of enamel rods was altered relative to controls. These results demonstrate distinct and varied spatiotemporal expression of alpha7 during tooth development, and they suggest that dysfunction of this receptor would have diverse impacts upon the adult organ