Vitamin D and its effects on glucose homeostasis, cardiovascular function and immune function.

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.

Recent trends and clinical features of childhood vitamin D deficiency presenting to a children's hospital in Glasgow.

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.

Changes in quantitative ultrasound in infants born at less than 32 weeks' gestation over the first 2 years of life: influence of clinical and biochemical changes.

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.

Quantitative ultrasound assessment of bone health in the neonate.

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.

Longitudinal changes in bone health as assessed by the speed of sound in very low birth weight preterm infants.

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.

Quantitative ultrasound assessment of bone in preterm and term neonates.

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.

A critical role for lymphotoxin-beta receptor in the development of diabetes in nonobese diabetic mice.

To assess the role of lymphotoxin-beta receptor (LTbetaR) in diabetes pathogenesis, we expressed an LTbetaR-Fc fusion protein in nonobese diabetic (NOD) mice. The fusion protein was expressed in the embryo, reached high levels for the first 2 wk after birth, and then declined progressively with age. High expression of LTbetaR-Fc blocked diabetes development but not insulitis. After the decline in chimeric protein concentration, mice became diabetic with kinetics similar to the controls. Early expression of fusion protein resulted in disrupted splenic architecture. However, primary follicles and follicular dendritic cells, but not marginal zones, developed in aged mice. Hence, LTbetaR signaling is required for diabetes development and regulates follicular and marginal zone structures via qualitatively or quantitatively distinct mechanisms.

A new model for rheumatoid arthritis?

A chance observation has led to the development of a new murine model for inflammatory arthritis. Arthritis is induced, and transferred, by T-cell-dependent antibodies to glucose-6-phosphate isomerase. This enzyme is expressed in all cells, and is detectable in serum. There are several similarities to rheumatoid arthritis (RA) in the murine disease. This elegant model raises several questions as to how and why a systemic response focuses inflammation so strongly on synovial joints. The model also re-introduces the possibility that antibodies to widely expressed self-proteins may play a role in the pathogenesis of RA.

Transgenic mice expressing surface markers for IFN-gamma and IL-4 producing cells.

The detection of T(H)1-type and T(H)2-type cells directly in situ would be of great value in the study of T(H) development and function in vivo. Transgenic mice expressing human Thy1 and mouse Thy1.1 under the control of the murine IFN-gamma and IL-4 promoters, respectively, have been generated. The hThy1(+) cells represent (with some temporal lag) most of the IFN-gamma-producing CD4(+) T-cells, while the mThy1.1(+) cells represent only a percentage of IL-4 secreting cells. This may be due to mono-allelic expression of the IFN-gamma and IL-4 genes. Since permeabilization is not required for the detection of the transgenic surface markers, these transgenic mice can facilitate the detection of T(H)1-type and T(H)2-type cells by flow cytometry with surface immunofluorescent staining. These surface markers should permit isolation of viable cells according to their T(H) type for adoptive transfer experiments, and may serve as a model system for tracing the development of T(H)1 and T(H)2-type cells in vivo.

Discovering the role of the major histocompatibility complex in the immune response.

The discovery that genes in the major histocompatibility complex (MHC) play an important role in the immune response depended on the chance interaction of several unrelated events. The first, and most important, was the decision by Michael Sela to synthesize a series of branched, multichain, synthetic polypeptides based on a backbone of poly-l-lysine. The prototype compound, (T,G)-A-L, was tipped with short random sequences of tyrosine and glutamic acid. This resulted in a restricted range of antigenic determinants composed of only two or three amino acids with a variable length-ideal for binding to the peptide binding groove of MHC class II molecules. The second was the decision by John Humphrey to immunize various strains of rabbits with this synthetic polypeptide. Two of these rabbit strains showed very large quantitative differences in antibody response to (T, G)-A-L. In transferring this system to inbred mouse strains, the third bit of good fortune was the availability at the National Institute of Medical Research, in Mill Hill (London), of the CBA (H2(k)) and C57 (H2(b)) strains. The H2(b) haplotype is the only one mediating a uniform high antibody response to (T,G)-A-L. The fourth critical ingredient was the availability of numerous congenic and H2 recombinant inbred strains of mice produced earlier by Snell, Stimpfling, Shreffler, and Klein. A search for congenic pairs of mice expressing the responder and nonresponder H2 haplotypes on the same background revealed that these strains responded as a function of their H2 haplotype, not of their inbred background. Extensive studies in a variety of inbred strains carrying recombinant H2 haplotypes, as well as a four-point linkage cross, mapped immune response to (T,G)A-L within the murine MHC, between the K and Ss loci. The demonstration that stimulation in the mixed lymphocyte reaction (MLR) mapped to the same region quickly led to attempts to produce antisera in congenic H2 recombinant strain combinations. These antisera identified I-region associated (Ia) antigens. Immunoprecipitation and blocking studies showed that the gene products controlling specific immune responses, the mixed lymphocyte reaction, and the structure of Ia antigens were one and the same-now designated as the I-A MHC class II molecules. These antisera and inbred strains enabled Unanue to demonstrate the peptide binding function of class II MHC molecules.

The role of MHC class II molecules in susceptibility to type I diabetes: identification of peptide epitopes and characterization of the T cell repertoire.

Susceptibility to type I diabetes is linked to class II MHC alleles in both mouse and man. However, the molecular mechanisms by which MHC molecules mediate disease susceptibility are unknown. To analyze how I-A alleles predispose to, or prevent, the development of type I diabetes, we have chosen, as the first step, to investigate the immune response to an important islet cell protein in diabetes-susceptible and diabetes-resistant mice. MHC class II alleles conferring susceptibility and resistance to diabetes select completely different sets of immunogenic epitopes from the beta islet cell autoantigen glutamic acid decarboxylase 65. Peptide-binding studies, analysis of MHC restriction, and immunization with these peptide epitopes indicate that the two amino acid substitutions within the I-A(beta) chain that distinguish a diabetes-susceptibility from a diabetes-resistance allele are sufficient to alter peptide binding and MHC restriction and may also influence antigen presentation and the selection of the T cell repertoire. The data indicate that the molecular mechanisms for class II-mediated selection of immunodominant epitopes are complex and differ for each individual peptide epitope. Further study of the functional characteristics of the response to these epitopes should provide insight into mechanisms of MHC-mediated diabetes susceptibility.

A small number of residues in the class II molecule I-Au confer the ability to bind the myelin basic protein peptide Ac1-11.

The N-terminal peptide Ac1-11 of myelin basic protein induces experimental autoimmune encephalomyelitis in H-2(u) and (H-2(u) x H-2(s)) mice but does not in H-2(s) mice. Ac1-11 binds weakly to the class II major histocompatibility complex (MHC) molecule I-Au but not at all to I-As. We have studied the interaction of Ac1-11 and I-Au as a model system for therapeutic intervention in the autoimmune response seen in experimental autoimmune encephalomyelitis. Two polymorphic residues that differ between I-Au and I-As, Y26beta and T28beta, and one conserved residue, E74beta, confer specific binding of Ac1-11 to I-Au. A fourth residue, R70beta in I-Au, affects both peptide binding and T cell recognition. These results are consistent with a model that places arginine at position five of Ac1-11 in pockets 4 and 7 of the MHC groove, which is formed in part by residues 26, 28, 70, and 74 of Abetau and places lysine at position four of Ac1-11, previously shown to be a major MHC contact, in hydrophobic pocket 6. The data indicate that the primary region of I-Au that confers specific binding of Ac1-11 lies in the center of the peptide binding groove rather than in the region that contacts the N terminus of the peptide, as has been shown for HLA DR and the homologous I-E molecules.

Rheumatoid arthritis and its animal models: the role of TNF-alpha and the possible absence of specific immune reactions.

Rheumatoid arthritis is an organ-specific inflammatory disease of humans. Recent studies have focused on associations with non-MHC genes, new autoantigens and the role of innate immune responses. The success of anti-TNF-alpha in the majority (but, interestingly, not all) of patients has implications for disease mechanisms but the dangers of long-term therapy are becoming clearer. A number of new models of arthritis have been defined and emphasize the importance of the genetic make-up of the host. Attention has also focused on why the joint is a particularly vulnerable site for inflammatory responses.

HLA class II transgenic mice: models of the human CD4+ T-cell immune response.

This review examines the field of current HLA class II transgenic mouse models and the individual approaches applied in production of these mice. The majority of these mice have been created with the objective of obtaining a disease model with clinical features mimicking human autoimmune disease. The development process of a different type of HLA class II transgenic mice, which are designed to function as a substitute for a normal human immune system in studies of human autoantigens, is described. Several HLA-DR4 transgenic lines with normally expressed HLA-DR4 molecules have been produced. To obtain adequate positive selection of the HLA-DR4-restricted CD4+ T-cell repertoire in these mice it is essential both to introduce a human CD4 transgene, and to delete the murine major histocompatibility complex (MHC) class II molecules. These HLA-DR4 transgenic mice have been used to determine the immunogenic CD4+ T-cell epitopes of several human autoantigenic proteins.

Identification of autoantigen epitopes in MHC class II transgenic mice.

MHC class II molecules function by selective binding of antigenic peptides, thereby both shaping the T-cell receptor (TCR) repertoire in the thymus and influencing presentation of immunogenic peptides to CD4+ T cells in the periphery. The strong association between a number of human autoimmune diseases (type 1 diabetes, rheumatoid arthritis, and multiple sclerosis) and certain HLA-DR/DQ alleles suggests that it may be possible to alter pathological autoimmune responses by deliberate introduction of autoantigenic peptides in a "tolerogenic" manner. Since there are likely to be differences in epitope selection and epitope spreading in different patients over time, this approach requires identification of all the immunogenic CD4+ T-cell epitopes (dominant, subdominant, or cryptic) of an autoantigen which elicit T-cell responses restricted to the HLA-DR/DQ alleles predisposing to these autoimmune diseases. This paper describes a new approach for the identification of immunogenic peptide epitopes of human autoantigenic proteins using HLA-DR and DQ transgenic mice. These mice are engineered to select a full TCR repertoire which can identify immunogenic peptide epitopes similar or identical to human subjects of the same HLA-DR/DQ genotype. This experimental system also allows comparison of autoantigenic immune responses restricted to disease-susceptible and disease-resistant HLA-DR/DQ alleles.

Prevention of diabetes in NOD mice by a mutated I-Ab transgene.

Susceptibility to the human autoimmune disease IDDM is strongly associated with those haplotypes of the major histocompatibility complex (MHC) carrying DQB1 alleles that do not encode aspartic acid at codon 57. Similarly, in a spontaneous animal model of this disease, the NOD mouse, the genes of the MHC play an important role in the development of diabetes. The DQB1 homolog in NOD mice, I-Ab(g7), encodes a histidine at codon 56 and a serine at codon 57, while all other known I-Ab alleles encode proline and aspartic acid, respectively, at these positions. We therefore mutated the NOD I-Ab allele to encode proline at position 56 and aspartic acid at position 57 and introduced this allele onto the NOD genetic background to study the effect of these substitutions on susceptibility to diabetes. No transgenic mice developed diabetes by 8 months of age, and transgenic mice had markedly reduced lymphocytic infiltration in the pancreas compared with nontransgenic littermates. Furthermore, splenocytes from transgenic mice failed to proliferate or secrete gamma-interferon in response to a panel of beta-cell autoantigens, although the mice did produce beta-cell specific antibodies. Interestingly, the proportion of IgG1 and IgE relative to IgG2a comprising these autoantibodies was much greater in transgenic mice compared with nontransgenic control mice. Finally, T-cells from transgenic mice inhibited the adoptive transfer of diabetes to irradiated recipients. This inhibition was partially reversed by treatment of the recipients with a combination of anti-interleukin (IL)-4 and anti-IL-10 monoclonal antibodies. Thus, a transgenic class II MHC allele encoding aspartic acid at B57 prevents diabetes, in part, by promoting the production of IL-4 and IL-10, which interfere with the effector phase of the diabetic process.