Autoantibodies and associated T-cell responses to determinants within the 831-860 region of the autoantigen IA-2 in Type 1 diabetes.

B-cells influence T-cell reactivity by facilitating antigen presentation, but the role of autoantibody-secreting B-cells in regulating T-cell responses in Type 1 diabetes is poorly defined. The aims of this study were to characterise epitopes on the IA-2 autoantigen for three monoclonal antibodies from diabetic patients by amino acid substitutions of selected residues of IA-2, establish contributions of these epitopes to binding of serum antibodies in Type 1 diabetes and relate B- and T-cell responses to overlapping determinants on IA-2. The monoclonal antibodies recognised overlapping epitopes, with residues within the 831-860 region of IA-2 contributing to binding; substitution of Glu836 inhibited binding of all three antibodies. Monoclonal antibody Fab fragments and substitution of residues within the 831-836 region blocked serum antibody binding to an IA-2 643-937 construct. IL-10-secreting T-cells responding to peptides within the 831-860 region were detected by cytokine-specific ELISPOT in diabetic patients and responses to 841-860 peptide were associated with antibodies to the region of IA-2 recognised by the monoclonal antibodies. The study identifies a region of IA-2 frequently recognised by antibodies in Type 1 diabetes and demonstrates that these responses are associated with T-cells secreting IL-10 in response to a neighbouring determinant.

Increased resistance to CD4+CD25hi regulatory T cell-mediated suppression in patients with type 1 diabetes.

Type I diabetes (T1D) is a T cell-mediated autoimmune disease characterized by loss of tolerance to islet autoantigens, leading to the destruction of insulin-producing beta cells. Peripheral tolerance to self is maintained in health through several regulatory mechanisms, including a population of CD4+CD25hi naturally occurring regulatory T cells (T(regs)), defects in which could contribute to loss of self-tolerance in patients with T1D. We have reported previously that near to T1D onset, patients demonstrate a reduced level of suppression by CD4+CD25hi T(regs) of autologous CD4+CD25- responder cells. Here we demonstrate that this defective regulation is also present in subjects with long-standing T1D (> 3 years duration; P = 0.009). No difference was observed in forkhead box P3 or CD127 expression on CD4+CD25hi T cells in patients with T1D that could account for this loss of suppression. Cross-over co-culture assays demonstrate a relative resistance to CD4+CD25hi T(reg)-mediated suppression within the CD4+CD25- T cells in all patients tested (P = 0.002), while there appears to be heterogeneity in the functional ability of CD4+CD25hi T(regs) from patients. In conclusion, this work demonstrates that defective regulation is a feature of T1D regardless of disease duration and that an impaired ability of responder T cells to be suppressed contributes to this defect.

T cell activation by coxsackievirus B4 antigens in type 1 diabetes mellitus: evidence for selective TCR Vbeta usage without superantigenic activity.

Numerous clinical and epidemiological studies link enteroviruses such as the Coxsackie virus group with the autoimmune disease type 1 diabetes mellitus (DM). In addition, there are reports that patients with type 1 DM are characterized by skewing of TCR Vbeta chain selection among peripheral blood and intraislet T lymphocytes. To examine these issues, we analyzed TCR Vbeta chain-specific up-regulation of the early T cell activation marker, CD69, on CD4 T cells after incubation with Coxsackievirus B4 (CVB4) Ags. CD4 T cells bearing the Vbeta chains 2, 7, and 8 were the most frequently activated by CVB4. Up-regulation of CD69 by different TCR families was significantly more frequent in new onset type 1 DM patients (p = 0.04), 100% of whom (n = 8) showed activation of CD4 T cells bearing Vbeta8, compared with 50% of control subjects (n = 8; p = 0.04). T cell proliferation after incubation with CVB4 Ags required live, nonfixed APCs, suggesting that the selective expansion of CD4 T cells with particular Vbeta chains resulted from conventional antigen processing and presentation rather than superantigen activity. Heteroduplex analysis of TCR Vbeta chain usage after CVB4 stimulation indicated a relatively polyclonal, rather than oligo- or monoclonal response to viral Ags. These results provide evidence that new-onset patients with type 1 DM and healthy controls are primed against CVB4, and that CD4 T cell responses to the virus have a selective TCR Vbeta chain usage which is driven by viral Ags rather than a superantigen.

Evidence for recognition of novel islet T cell antigens by granule-specific T cell lines from new onset type 1 diabetic patients.

Type 1 diabetes is a T cell-mediated autoimmune disease where a number of islet beta-cell target autoantigens have been characterized on the basis of reactivity with autoantibodies. Nevertheless, there remains uncertainty of the nature of another group of autoantigens associated with the secretory granule fraction of islet beta-cells that appear to be targeted predominantly by autoreactive T cells. We have previously characterized CD4+, HLA-DR-restricted T cell lines from new onset type 1 diabetic patients that are specific for the secretory granule fraction of rat tumour insulinoma, RIN. The T cell line from the first patient, HS, proliferates in response to crude microsomal membranes prepared from a recently established, pure human islet beta-cell line NES2Y. In addition, the HS line also responds to secretory granule fractions prepared from a murine tumour insulinoma grown in RIP-Tag mice, showing the recognition of species-conserved antigen(s) in beta-cells. Using partially matched antigen-presenting cells, the HS T cells and another line derived from a second patient, MR, were shown to be restricted by disease-associated DRB1*0101 and DRB1*0404 alleles, respectively. Neither the HS or MR T cell lines proliferate in response to a large panel of candidate islet cell antigens, including insulin, proinsulin, glutamic acid decarboxylase, the protein tyrosine phosphatase IA-2/phogrin, imogen-38, ICA69 or hsp60. Our data provide compelling evidence of the presence of a group of antigens associated with the secretory granule fraction of islet beta-cells recognized by the T cell lines, whose definition may contribute to our knowledge of disease induction as well as to diagnosis.

Cushing's syndrome associated with a chemodectoma and a carcinoid tumour.

We present a case of Cushing's syndrome where 111In-octreotide scanning provided evidence for the presence of two neuroendocrine tumours. Uptake in the right neck corresponded to a chemodectoma, but there was no change in the clinical condition or fall in ACTH levels following surgical resection. Uptake in the left chest was assumed to relate to a bronchial carcinoid, but a tumour could not initially be localized on magnetic resonance imaging (MRI), spiral CT scanning or on selective venous sampling. A 1 cm bronchial carcinoid tumour was identified post-mortem which immunostained for ACTH. This case demonstrates that 111ln-octreotide scanning is a useful technique for identifying the source of ectopic ACTH production in difficult cases of Cushing's syndrome. Reliance should not be placed solely on standard imaging techniques to localize the tumour prior to surgery. Although rare, the possibility of a non-ACTH secreting neuroendocrine tumour should also be considered in patients with ectopic ACTH syndrome, who have positive 111In-octreotide scans.

Comparative analysis of epitope recognition of glutamic acid decarboxylase (GAD) by autoantibodies from different autoimmune disorders.

Autoantibodies to GAD, an important marker of the autoimmune process in type I or insulin-dependent diabetes mellitus (IDDM), are also found in non-diabetic individuals with autoimmune polyendocrine syndrome type 1 (APS1), APS2, and stiff man syndrome (SMS). Most IDDM sera contain two distinct GAD antibody specificities, one of which targets an epitope region in the middle-third of GAD65 (IDDM-E1; amino acids 221-359) and one of which targets the carboxy-third of GAD65 (IDDM-E2; amino acids 453-569). Using 11 chimeric GAD65/GAD67 proteins to maintain conformation-dependent epitopes of GAD65, we compared the humoral repertoire of IgG antibodies from an individual with APS2-like disease (b35, b78, and b96) and MoAbs from an IDDM patient (MICA-2, MICA-3, and MICA-4). Neither the APS2 IgG antibodies nor the IDDM MoAbs bind the amino-terminal third of GAD65, but instead target the carboxy-terminal two-thirds of GAD65. Amino acids 270-359 (IDDM-E1) are targeted by one APS2 IgG antibody and MICA-4, while two other APS2 IgG antibodies, MICA-2 and MICA-3, target amino acids 443-585 (IDDM-E2). Using GAD65/67 chimera that span the IDDM-E2 region, we found that MICA-2 binds amino acids 514-528 of GAD65, but two APS2 IgG antibodies require this region and amino acids 529-570. In contrast, the binding of MICA-3 requires two discontinuous amino acid segments of GAD65 (452-513 and 528-569), but not amino acids 514-528. These results indicate that there are both similarities and differences in the humoral response to GAD65 in APS2 and IDDM.

Is continued weight gain inevitable in type 2 diabetes mellitus?

Prevention and treatment of obesity are major clinical problems encountered in the management of Type 2 diabetes mellitus (DM); indeed, up to 90% of such patients are regarded as being overweight. Except for a brief period following diagnosis, when presumably enthusiasm to adopt lifestyle change is at its greatest, weight gain is generally progressive unless severe hyperglycaemia or complications intervene. Even a relatively modest weight loss of 10% can have major benefits in terms not only of reducing the risk of developing DM in the first place, but also in improving metabolic control after the disorder has become established. Behavioural therapy (BT) in combination with hypocaloric diet achieves weight loss in the short-term, but is poorly sustained in the long-term. Exercise has metabolic benefits beyond its rather minimal effects on short-term weight loss in that it may also aid long-term weight control. The difficulties encountered in maintaining lifestyle change do, however, suggest the need for ongoing intervention--perhaps including a regular period on a stricter dietary regimen (800-1000 kcalday-1), possibly a very low calorie diet (VLCD)(< 800 kcalday-1) or even the use of orlistat, a pancreatic lipase inhibitor which reduces the absorption of dietary fat. Realistically, the aim should be for long-term weight stability.

Human B cells secreting immunoglobulin G to glutamic acid decarboxylase-65 from a nondiabetic patient with multiple autoantibodies and Graves' disease: a comparison with those present in type 1 diabetes.

Antibodies to glutamic acid decarboxylase-65 (GAD65) are present in a number of autoimmune disorders, such as insulin-dependent (type 1) diabetes mellitus (IDDM), stiff man syndrome, and polyendocrine autoimmune disease. Antibodies to GAD in IDDM patients usually recognize conformation-dependent regions on GAD65 and rarely bind to the second isoform, glutamic acid decarboxylase-67 (GAD67). In contrast, those present in stiff man syndrome and polyendocrine disease commonly target the second isoform (GAD67) and include antibodies that are less dependent on the conformation of the molecule. By immortalizing peripheral blood B cells with Epstein-Barr virus, we have generated three human IgG autoantibodies, termed b35, b78, and b96, to GAD65 from one patient with multiple autoantibodies to endocrine organs and Graves' disease. All three autoantibodies are of the IgG1 isotype, with islet cell activity, and do not react with GAD67. The regions on GAD65 recognized by the three autoantibodies have been investigated by immunoprecipitation with a series of chimeras, by binding to denatured and reduced antigens, and using protein footprinting techniques. Using chimeric GAD proteins, we have shown that b35 targets the IDDM-E1 region of GAD65 (amino acids 240-435) whereas both b78 and b96 target the IDDM-E2 region of GAD65 (amino acids 451-570). Furthermore, examination of binding to recombinant GAD65 and GAD67 by Western blotting revealed some differences in epitope recognition, where only b78 bound denatured and reduced GAD65. However, b35, b78, and b96 autoantibodies had different footprinting patterns after trypsin treatment of immune complexes with GAD65, again indicating different epitope recognition. Our results indicate that antibodies to GAD65 present in nondiabetic patients with multiple autoantibodies to endocrine organs show similarities to those in IDDM (by targeting IDDM-E1 and IDDM-E2 regions of GAD65) as well as subtle differences in epitope recognition (such as binding to denatured and reduced GAD65 and by protein footprinting). Thus, the GAD65 epitopes recognized by autoantibodies in different autoimmune diseases may overlap and be more heterogeneous than previously recognized.

Birthweight for length: ponderal index, body mass index or Benn index?

This study compares how effectively the ponderal index and the body mass index adjust birthweight for length at different gestations, and derives an improved index suitable for all gestations. The study was a cross-sectional survey, in a London teaching hospital, using a total of 999 neonates of 33 weeks gestation or later. Main outcome measures were the ponderal index (birthweight/length3), body mass index (birthweight/length2), and Benn index (birthweight/length(n)), where the length power n varies with gestation and is estimated by log-log regression. Results showed that up to 39 weeks gestation, the ponderal index is uncorrelated with length and so is a good index of birthweight for length. Past 39 weeks gestation, the ponderal index is negatively correlated with length, while the body mass index is uncorrelated, so that the body mass index is better. Neither index is optimal at all gestations. Deriving the Benn index (birthweight/length(n)) for each week of gestation, choosing n to make the index uncorrelated with length, shows that n falls steadily and very significantly (p < 0.0001) with increasing gestation. This in turn means that predicted birthweight for length depends on gestation: for a neonate 48 cm long, predicted birthweight varies from 2485 g at 34 weeks to 3030 g at 43 weeks, a 20% range. However, for a 54 cm long infant, predicted birthweight is the same at all gestations. A Benn index where the value of n changes linearly with gestation is described. We conclude that the ponderal index is not appropriate for measuring intra-uterine malnutrition, as it fails to adjust for length at all gestations. No other index of birthweight/length(n) with constant n is any better, as different gestations require different indices. Birthweight predicted from an infant's length depends on the infant's gestation. If, as Barker proposes, thinness at birth assessed by birthweight for length is used to predict later health status, more account needs to be taken of the complex relationship between birthweight, length and gestation.

Human immunoglobulin G autoantibodies to the thyrotropin receptor from Epstein-Barr virus-transformed B lymphocytes: characterization by immunoprecipitation with recombinant antigen and biological activity.

The TSH receptor (TSH-R) is the target antigen for disease-related autoantibodies in Graves' disease and primary myxoedema, but the repertoire of the antibodies or the nature of the precise antigenic epitopes is not known. We have immortalized peripheral blood B cells from six different autoimmune thyroid disease patients with Epstein-Barr virus and selected IgG-producing B cells by magnetic selection on anti-IgG-coated beads. Purified recombinant insect cell-derived extracellular region of TSH-R was used to identify the positive wells for expansion in culture. Stable B cell lines (n = 11) were obtained, which after limiting dilution led to two stable B cell clones. B cell lines and clones secreted IgG antibody that were shown to react biochemically with metabolically labeled or in vitro translated, nascent extracellular region of TSH-R, giving strong, confirmatory evidence of the presence of anti-TSH-R antibody. Supernatants from lines contained thyroid-stimulating activity, thyroid-blocking activity (as assessed by inhibition of TSH-mediated cAMP stimulation), or both of these activities. Interestingly, antibodies with stimulating activity were generated from a primary myxoedema patient, and antibodies of blocking specificities were obtained from newly diagnosed thyrotoxic Graves' disease patients. Our results favor a fine balance between stimulating and blocking autoantibody activities in determining the clinical presentation observed in patients with autoimmune thyroid disease patients who have these antibodies present in their serum.

Binding of antithyrotropin receptor autoantibodies in Graves' disease serum to nascent, in vitro translated thyrotropin receptor: ability to map epitopes recognized by antibodies.

The binding of Graves' disease autoantibodies and xenogeneic antibodies to the human TSH receptor (TSH-R) has been studied using receptor preparations generated in an in vitro transcription and translation reaction. The complementary DNAs encoding for the full-length (764 amino acids) and the extracellular region of TSH-R (amino acids 20-414, lacking the signal sequence) were used to generate the translated receptor antigen. Stable [35S]methionine-labeled nascent protein for full-length and extracellular regions of TSH-R of approximate size 87 and 50 kDa, respectively, together with other smaller proteins were generated. The 87- and 50-kDa translated receptor proteins react by immunoprecipitation analysis with monoclonal antibodies, polyclonal antisera, and unfractionated Graves' disease serum containing autoantibody to TSH-R. The translated products of the extracellular TSH-R were examined in detail. Using three well characterized murine monoclonal antibodies whose epitopes encompass the amino-, central, and carboxyl-terminals of the extracellular region of the receptor led to immunochemical identification of the smaller translated products to derive from internal methionine start sites of TSH-R. These smaller, N-terminal-truncated translated proteins were also recognized by polyclonal antisera generated against recombinant TSH-R, thus allowing epitope mapping of some antibodies. A large proportion of Graves' disease autoantibodies (> 70%) bind to the translated extracellular region of TSH-R. This indicates that the majority of pathogenic anti-TSH-R autoantibody binds the nascent translated extracellular region of TSH-R, which is not influenced by the lack of glycosylation of the receptor.

HLA-DR-restricted T cell lines from newly diagnosed type 1 diabetic patients specific for insulinoma and normal islet beta cell proteins: lack of reactivity to glutamic acid decarboxylase.

T cells reacting with pancreatic islet beta cell proteins play a pivotal role in the pathogenesis of type 1 diabetes in experimental animal models and man, although the islet cell autoantigens against which these T cells are directed remain to be characterized. We have previously shown the presence of disease-related antigens residing in the transplantable RIN insulinoma membranes which are recognized by T cells from diabetic NOD mice. We now report on the establishment of CD4+, T cell lines reacting with insulinoma membranes from six newly diagnosed type 1 diabetic patients. Detailed examination of T cell lines from two patients revealed that both the lines continued to react with normal islet cell proteins and, interestingly, were also stimulated by antigens present in brain microsomes. The two T cell lines showed reactivity with different molecular weight proteins of the insulinoma membranes and both the lines were histocompatibility-linked antigen (HLA)-DR restricted. Although the insulinoma membrane preparation is known to contain glutamic acid decarboxylase (GAD), none of the six T cell lines proliferates in response to purified GAD. These T cell lines will be valuable in characterizing novel islet beta cell antigens which are likely to be implicated in type 1 diabetes.

Head circumference/abdominal circumference ratio, ponderal index and fetal malnutrition. Should head circumference/abdominal circumference ratio be abandoned?

Head circumference/abdominal circumference (HC/AC) ratios of the fetus are accepted as a means of distinguishing different patterns of growth retardation with a high ratio implying malnutrition of the fetus. Ponderal index (birthweight/length3) is used by paediatricians as a measure of neonatal wasting and would therefore be expected to correlate with HC/AC ratios at delivery. Anthropometric data on 999 newborn infants have been collected and analyzed by multiple regression. The results show a poor correlation between ponderal index and HC/AC ratio, worse than that between ponderal index and AC alone. The use of HC/AC ratios antenatally to identify subgroups of intrauterine malnutrition should be abandoned. The prediction of intrauterine malnutrition by weight/length ratios should be investigated further.