Dermatologic and Ocular DiseasesReduced soluble CD14 levels in amniotic fluid and breast milk are associated with the subsequent development of atopy, eczema, or both☆,☆☆
Section snippets
Source of samples
Samples from discrete cohorts were used, and these are summarized in Table I.
Study number* Samples Age at clinical assessment† Complete or ongoing Study 1 Serial plasma at birth (cord blood), 6 mo, 1 y, and 5 y of age; high-risk cohort 6 mo and 1, 2, 3, 4, and 5 y Complete Study 2 Amniotic fluid at diagnostic amniocentesis (16-18 wk of gestation); not selected on family history of atopy 1 y Ongoing Study 3 Breast milk at 3 mo postpartum;
RT-PCR
RNA was extracted by using RNase-free DNase treatment with the RNeasy total RNA isolation system, as directed by the manufacturer (Qiagen Ltd). First-strand cDNA synthesis was performed with 0.5 μg of total RNA in a 20-μL reaction by using the Omni-script reverse transcriptase kit (Qiagen Ltd) primed with oligo T17 (AGC, Sigma Genosys), as recommended by the manufacturer.
PCR reactions were performed in a total reaction volume of 25 μL with 1 μL of cDNA in 1× reaction buffer, with 1.5 mmol/L MgCl
Maturation of sCD14 levels
Samples from studies 4 and 5 were used to examine the natural maturation of circulating sCD14 levels. Fetal-neonatal plasma (study 5) sCD14 levels increased significantly with gestational age but remained significantly lower at term than those in adults (Fig 1, A ).
Discussion
To study the potential contribution of sCD14 to the development of atopic diseases in infancy, we took advantage of various samples from a number of cohorts collected by our group over the past few years. One of these cohorts (study 1) has completed clinical assessment, whereas the remainder (studies 2, 3, and 4) are ongoing. Differences in the timing of clinical assessment referred to throughout reflect the different ages of the cohort being analyzed. Soluble CD14 was the focus of this study
Acknowledgements
We thank the staff in the Department of Fetal Medicine and in the delivery suite at the Princess Anne Hospital, Southampton, for their assistance in the collection of samples and to the MRC Tissue Bank for fetal tissue. We also thank all the mothers and children who participated in this study.
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Supported by the National Heart, Lung, and Blood Institute (grant number HL61858); the National Asthma Campaign (UK); the British Lung Foundation; and the Food Standards Agency (grant number T07005).
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Reprint requests: Catherine A. Jones, PhD, Allergy and Inflammation Sciences, Level G Mailpoint 803, Southampton General Hospital, Southampton, UK, SO16 6YD.