Background: Between 1961-1971 vitamin D deficiency was recognized as a public health issue in the UK, because of the lack of effective sunlight and the population mix [1, 2]. In recent years, health care professionals have cited evidence suggesting a re-emergence of the vitamin D deficiency linked to a number of health consequences as a concern [3-6]. Evidence from observational studies has linked low vitamin D status with impairment in glucose homeostasis and immune dysfunction [7-9]. However, interventional studies, particularly those focused on paediatric populations, have been limited and inconsistent. There is a need for detailed studies, to clarify the therapeutic benefits of vitamin D in these important clinical areas. Objective: The aims of this PhD thesis were two-fold. Firstly, to perform preliminary work assessing the association between vitamin D deficiency and bone status, glucose homeostasis and immune function, and to explore any changes in these parameters following short term vitamin D3 replacement therapy. Secondly, to assess the effectiveness of an electronic surveillance system (ScotPSU) as a tool to determine the current incidence of hospital-based presentation of childhood vitamin D deficiency in Scotland. Methods: Active surveillance was performed for a period of two years as a part of an electronic web-based surveillance programme performed by the Scottish Paediatric Surveillance Unit (ScotPSU). The validity of the system was assessed by identifying cases with profound vitamin D deficiency (in Glasgow and Edinburgh) from the regional laboratory. All clinical details were checked against those identified using the surveillance system. Thirty-seven children aged 3 months to 10 years, who had been diagnosed with vitamin D deficiency, were recruited for the bone, glucose and immunity studies over a period of 24 months. Twenty-five samples were analysed for the glucose and bone studies; of these, 18 samples were further analysed for immune study. Treatment consisted of six weeks taking 5000 IU units cholecalciferol orally once a day. At baseline and after completion of treatment, 25 hydroxyvitamin D (25(OH)D), parathyroid hormone (PTH), alkaline phosphatase (ALP), collagen type 1 cross-linked C-telopeptide (CTX), osteocalcin (OCN), calcium, phosphate, insulin, glucose, homeostasis model assessment index, estimated insulin resistance (HOMA IR), glycated hemoglobin (HbA1c), sex hormone binding globulin (SHBG), lipids profiles, T helper 1 (Th1) cytokines (interleukin-2 ( IL-2), tumor necrosis factors-alpha (TNF-α), interferon-gamma (INF-γ)), T helper 2 (Th2) cytokines (interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6)), T helper 17 (Th17) cytokine (interleukin-17 (IL-17)), Regulatory T (Treg) cytokine (interleukin-10 (IL-10)) and chemokines/cytokines, linked with Th1/Th2 subset balance and/or differentiation (interleukin-8 (IL-8), interleukin-12 (IL-12), eosinophil chemotactic protein ( EOTAXIN), macrophage inflammatory proteins-1beta (MIP-1β), interferon-gamma-induced protein-10 (IP-10), regulated on activation, normal T cell expressed and secreted (RANTES), monocyte chemoattractant protein-1(MCP-1)) were measured. Leukoocyte subset analysis was performed for T cells, B cells and T regulatory cells and a luminex assay was used to measure the cytokiens. Results: Between September 2009 and August 2011, 163 cases of vitamin D deficiency were brought to the attention of the ScotPSU, and the majority of cases (n = 82) were reported in Glasgow. The cross-validation checking in Glasgow and Edinburgh over a one-year period revealed only 3 (11%) cases of clearly symptomatic vitamin D deficiency, which had been missed by the ScotPSU survey in Glasgow. While 16 (67%) symptomatic cases had failed to be reported through the ScotPSU survey in Edinburgh. For the 23 children who are included in bone and glucose studies, 22 (96%) children had basal serum 25(OH)D in the deficiency range ( 50-75 nmol/l, 12 (52%) children reached a final serum 25(OH)D >75-150 nmol/l and finally 3 (13%) exceeded the normal reference range with a final 25(OH)D >150 nmol/l. Markers for remodelling ALP and PTH had significantly decreased (p = 0.001 and <0.0001 for ALP and PTH respectively). In 17 patients for whom insulin and HOMA IR data were available and enrolled in glucose study, significant improvements in insulin resistance (p = 0.04) with a trend toward a reduction in serum insulin (p = 0.05) was observed. Of those 14 children who had their cytokines profile data analysed and enrolled in the immunity study, insulin and HOMA IR data were missed in one child. A significant increase in the main Th2 secreted cytokine IL-4 (p = 0.001) and a tendency for significant increases in other Th2 secreted cytokines IL-5 (p = 0.05) and IL-6 (p = 0.05) was observed following vitamin D3 supplementation. Conclusion: An electronic surveillance system can provide data for studying the epidemiology of vitamin D deficiency. However, it may underestimate the number of positive cases. Improving vitamin D status in vitamin D deficient otherwise healthy children significantly improved their vitamin D deficient status, and was associated with an improvement in bone profile, improvements in insulin resistance and an alteration in main Th2 secreting cytokines.

In recent years there has been increasing interest in the non-skeletal effects of vitamin D. It has been suggested that vitamin D deficiency may influence the development of diabetes, cardiovascular dysfunction and autoimmune diseases. This review focuses on the current knowledge of the effects of vitamin D and its deficiency on cardiovascular function, glucose homeostasis and immune function, with a particular focus on children. Although, there is good evidence to show that there is an association between vitamin D deficiency and an abnormality of the above systems, there is little evidence to show that vitamin D supplementation leads to an improvement in function, especially in childhood.

BACKGROUND:The incidence of vitamin D deficiency is unclear in the context of continuing demographic changes and the introduction of new public health measures.METHODS:All cases in which vitamin D deficiency was suspected as the primary cause of the clinical presentation were studied.RESULTS:Between 2002 and 2008, 160 cases of symptomatic vitamin D deficiency were identified with twice as many cases in 2008 (n, 42) as in the previous years. The median age of the cohort was 24 months (range 2 weeks-14 years).Three cases were recorded in children of European background, whereas the rest were in children of South Asian, Middle Eastern or sub-Saharan ethnic background. Presenting features included bowed legs in 64 (40%) and a fit in 19 (12%). In one infant, concerns were raised following a presentation with cardiac failure and hypocalcaemia.SUMMARY:Symptomatic vitamin D deficiency remains prevalent in the West of Scotland. There is a need for effective public health education, action and surveillance.

Studies in preterm infants show reduced speed of sound (SOS) as measured by quantitative ultrasound (QUS) during the immediate neonatal period. There is a scarcity of data on SOS changes following hospital discharge. The aim of this study was to assess SOS over the first 2 years in preterm infants. Infants were recruited from a neonatal follow-up clinic. Tibial QUS was performed using the Omnisense 7000P scanner. Thirty-nine infants born at < 32 weeks' gestation had a single SOS measurement (median 3,203 m/second, range 2,609-3,495) which correlated with corrected gestational age (CGA) (r = 0.8, P < 0.005). The majority of measurements were within the manufacturer's reference range for term infants. SOS standard deviation score (SDS) in infants aged 0-6 months CGA demonstrated a negative correlation with duration of total parenteral nutrition (r = 0.7, P < 0.05) and a positive correlation with serum phosphate (r = 0.6, P < 0.05.) Two groups of infants had serial measurements: eight had measurements performed at term CGA and early infancy (early) and seven had measurements in later infancy (late). In the early group SOS SDS increased (P < 0.005), and the greatest increase in SOS over time occurred in infants with the lowest SOS at term (r = 0.9). In the late group there was no significant change over time. SOS SDS change did not show any correlation to weight SDS change. Catch-up in SOS occurs postterm in most infants by 6 months and is independent of postnatal growth. Infants with the lowest SOS at term have the fastest rate of catch-up. The opportunity for catch-up may be greatest in early infancy.

For a number of reasons there is a need to explore reliable non-invasive methods for assessing bone health in neonates and young infants. Epidemiological studies suggest that early events in life may predispose the adult to degenerative diseases such as osteoporosis. Preterm infants have an increased risk of low bone mass because of limited bone mass accretion in utero and a greater need for bone nutrients. Despite improvements in neonatal care fractures still occur. The diagnosis of osteopaenia of prematurity remains difficult as there is no screening test which is both sensitive and specific. Biochemical indices are non-diagnostic, and plain X-rays in the absence of fractures are poor at diagnosing bone disease. Although dual energy X-ray absorptiometry is increasingly used to assess bone mineral status in newborn infants, the size and immobility of the scanner, the length of time to perform the scan and use of ionising radiation make it unsuitable for routine use in the setting of the fragile very low birth weight infant. Quantitative ultrasound (QUS) was first developed in 1984, as a non-ionising, portable and low cost method of assessing bone health. The measurements obtained from QUS are thought to be related not only to the mineral density of the bone but also to reflect parameters of bone quality and strength. Preliminary studies suggest that this technique may be a useful method of assessing changes in bone health in preterm infants, but the data need to be interpreted carefully. This review will concentrate on the methodology of QUS and the studies that have already been performed in neonates.

OBJECTIVE:To assess longitudinal changes in speed of sound (SOS) in very low birth weight (VLBW) infants and investigate the relationship with markers of osteopathy of prematurity (OP) and clinical illness. STUDY DESIGN:Twenty-five infants were recruited. Eighteen infants, median gestation 27 weeks (range 24-32), median birth weight 957 g (range 625-1500 g), had serial scans. SOS was measured at both tibiae weekly until 35 to 37 weeks corrected gestational age (CGA). RESULTS:Initial median SOS standard deviation score (SDS) (Z) score was -0.07 (range-1.3-1.3). SOS correlated with gestation (r, 0.8, P<.005), and birth weight (r, 0.67, P<.005.) SOS fell from a median of 2923 m/s (2672-3107) at birth to 2802 m/s (2502-2991) at 35 to 37 weeks CGA (P<.05). This fall was greater in the 24- to 27-week gestation cohort with a median reduction of 2.2 SDS (1.6, 4.0) compared with 1.3 SDS (0.8-2.2) in those>28 weeks (P<.05). There was a negative correlation between SOS, at the end of the study, peak serum alkaline phosphatase (ALP) (r, 0.6, P<.05), CRIB (Clinical Risk Index for Babies)/CRIB II scores (both r, 0.6, P<.05), and duration of total parenteral nutrition (TPN) (r, 0.58, P<.05.) CONCLUSIONS:Although tibial SOS was within the expected range at birth, there was a subsequent failure to gain SOS, and this was most marked in infants of a lower gestation.

There is a need to explore novel methods of assessing bone health in sick preterm infants. This study of the speed of sound in the long bones of newborn term and preterm infants shows that, in this population, this technique is not site specific and has a high degree of interobserver and intraobserver precision. The speed of sound in newborn infants is primarily dependent on gestation rather than birth weight.

TNF- (cid:1) has been linked to the development of type 1 diabetes (T1D). We previously reported that neonatal treatment of nonobese diabetic (NOD) mice with TNF- (cid:1) accelerated the onset of T1D, whereas TNF- (cid:1) blockade in the same time period resulted in a complete absence of diabetes. The mechanisms by which TNF- (cid:1) modulates development of T1D in NOD mice remain unclear. Here we tested the effects of TNF- (cid:1) on the maturation of dendritic cells (DCs) in the NOD mouse. We found that neonatal treatment with TNF- (cid:1) caused an increase in expression of maturation markers on CD11c (cid:2) CD11b (cid:2) DC subpopulations, whereas treatment with anti-TNF- (cid:1) resulted in a decrease in expression of maturation markers in the CD11c (cid:2) CD11b (cid:2) subset. Moreover, neonatal treatment with TNF- (cid:1) resulted in skewed development of a CD8 (cid:1) (cid:2) CD11b (cid:3) CD11c (cid:2) DC subset such that TNF- (cid:1) decreases the CD8 (cid:1) (cid:2) CD11c (cid:2) DC subset, increases the CD11c (cid:2) CD11b (cid:2) subset, and causes an increase in the expression of CD40 and CD54 on mature DCs capable of inducing immunity. Anti-TNF- (cid:1) -treated mice had an increase in the CD8 (cid:1) (cid:2) CD11c (cid:2) DCs. Notably, adoptively transferred naı¨ve CD4 (cid:2) T cells from BDC2.5 T cell receptor transgenic mice proliferated in the pancreatic lymph nodes in TNF- (cid:1) -treated NOD mice but not in anti-TNF- (cid:1) -treated mice. Finally, we show that anti-TNF- (cid:1) -treated mice showed immunological tolerance to islet cell proteins. We conclude that TNF- (cid:1) plays an important role in the initiation of T1D in the NOD mouse by regulating the maturation of DCs and, thus, the activation of islet-specific pancreatic lymph node T cells. In the present study, we hypothesized that the effects of TNF- (cid:2) and anti-TNF- (cid:2) in the neonatal period are mediated through the effects of TNF- (cid:2) in activating, and anti-TNF- (cid:2) in preventing, the maturation of DCs in the islets and pancreatic lymph nodes (PLNs). Here, we have explored possible mechanisms underlying the increase and decrease of disease activity by neonatal TNF- (cid:2) and anti-TNF- (cid:2) treatment, respectively, in wild-type NOD mice. We monitored DC maturation in PLNs and other lymph nodes (LNs) in NOD mice treated with TNF- (cid:2) and anti-TNF- (cid:2) and examined the impact on the activation of a pathogenic T cell response in an adoptive transfer model using BDC2.5 TCR tg T cells. Finally, we tested whether neonatal treatment with anti-TNF- (cid:2) renders animals partially or almost completely immunologically tolerant. Our data indicate that, by keeping DCs immature, anti-TNF- (cid:2) induces immunologic tolerance to islet cell proteins whereas TNF- (cid:2) induces DC maturation, resulting in an increased immune response. We conclude that TNF- (cid:2) plays a crucial role in the initiation of T1DM by modulating DC–T cell interactions via modulation of DC development.

CD4+CD25+Foxp3+ regulatory T cells (Treg) are critical for controlling autoimmunity. Evidence suggests that Treg development, peripheral maintenance, and suppressive function are dependent on Ag specificity. However, there is little direct evidence that the Treg responsible for controlling autoimmunity in NOD mice or other natural settings are Ag specific. In fact, some investigators have argued that polyclonal Ag-nonspecific Treg are efficient regulators of immunity. Thus, the goal of this study was to identify, expand, and characterize islet Ag-specific Treg in NOD mice. Ag-specific Treg from NOD mice were efficiently expanded in vitro using IL-2 and beads coated with recombinant islet peptide mimic-MHC class II and anti-CD28 mAb. The expanded Ag-specific Treg expressed prototypic surface markers and cytokines. Although activated in an Ag-specific fashion, the expanded Treg were capable of bystander suppression both in vitro and in vivo. Importantly, the islet peptide mimic-specific Treg were more efficient than polyclonal Treg in suppressing autoimmune diabetes. These results provide a direct demonstration of the presence of autoantigen-specific Treg in the natural setting that can be applied as therapeutics for organ-specific autoimmunity.

Summary: Immunization of rabbits, and inbred strains of mice with branched, multichain, synthetic polypeptides, such as (T, G) − A‐‐L and (H, G) − A‐‐L, revealed striking differences in the ability of different strains of mice to produce specific antibody. F1 and F1 × parental backcross mice revealed clear genetic control. Initial attempts to link this genetic control to known genetic markers were unsuccessful. The second approach, which attempted to transfer response from high or immediate responders into low responder recipients, initially encountered graft vs. host and host vs. graft reactions. The transfer of F1 spleen cells into the low responder parent demonstrated that ability to respond was a property of immunocompetent cells (spleen cells), not of the recipient's background genes. Mapping studies with recombinant H2 haplotype congenic strains, and a classic 4‐point mapping cross were concordant in placing the gene controlling this trait within the H2 complex, between the K and Ss loci. Subsequent studies mapped the genes for stimulation in the mixed lymphocyte culture reaction to the same position, suggesting cell surface expression. Production of antisera to ‘I‐region’ products defined ‘Ia’ antigens, the 2‐chain α/β MHC class II molecules.