Biomedical subjects

A Lernmark

Publications and source records attributed to A Lernmark.

At least 127 records · Page 7Linked to original sources

PubMed1996

A comparison of three statistical models for IDDM associations with HLA.

The association between HLA-DQ haplotypes and insulin-dependent diabetes mellitus (IDDM) was studied in 48 children from 44 families ascertained from the high incidence area around Umeå, Sweden. Numerous hypotheses have been proposed to explain associations between HLA and IDDM, but comparisons of statistical models based on these hypotheses have not been attempted. The aim of the present study was to compare the goodness-of-fit and predictive abilities among different statistical models. A likelihood-based analysis rather than a conventional analysis based on contingency tables was therefore adopted. We first used parental haplotype information in a conditional likelihood analysis (1) and then compared this analysis with that of an unaffected control group which used information on geographically matched controls. Under the analysis conditional on parental haplotype, a statistical model motivated by the hypothesis that the entire DQ heterodimer is involved in IDDM pathogenesis fit the data significantly better and had greater predictive ability than either a model motivated by the explanation that an IDDM gene is linked to DQB1 or that the DQB1 chain itself is involved in IDDM pathogenesis, or a model arising from the hypothesis that single amino acids at codon 57 of DQB1 and codon 52 of DQA1, respectively, confer susceptibility. Under the case-control analysis, the identity of the best-fitting or more predictive statistical model was not as clear, although both approaches to analyzing risk suggested that the single-amino-acids model had significantly poorer fit compared to the remaining two models.

Diabetes Mellitus, Type 1↗

PubMed1996

Antibodies to glutamic acid decarboxylase and insulin-dependent diabetes in patients with autoimmune polyendocrine syndrome type I.

To evaluate the association of autoimmunity to glutamic acid decarboxylase (GAD) with insulin-dependent diabetes mellitus (IDDM) and IDDM-associated human leukocyte antigen (HLA) types, we studied a unique group of 47 patients with autoimmune polyendocrine syndrome type 1, a recessive disease not associated with HLA. GAD65 antibodies (GAD65-Ab), GAD67-Ab, islet cell antibodies, and HLA-DQA1, -DQB1, and -DRB1 were analyzed in relation to IDDM or a decreased insulin secretory capacity. GAD65-Ab were found in six of the eight diabetic patients 0.9-8.0 yr before the onset of IDDM and in 16 (41%) nondiabetic patients during a follow-up of 2.4-19.5 yr. Eleven (28%) nondiabetic patients had GAD67-Ab and islet cell antibodies. Fasting C peptide (mean +/- SD, 0.5 +/- 0.24 vs. 1.03 +/- 0.49 nmol/L; P = 0.003) and first phase insulin response (75.6 +/- 37.9 vs. 166.4 +/- 112.7 mU/L; P = 0.019) were lower in patients with than in those without GAD65-Ab. No HLA genotype predominated in the IDDM patients or GAD65-Ab-positive nondiabetic patients, but the IDDM high risk genotypes were decreased in frequency among the patients with GAD65-Ab. In conclusion, nondiabetic autoimmune polyendocrine syndrome type 1 patients frequently have GAD65-Ab together with a decreased insulin secretory capacity, suggesting subclinical islet cell inflammation not invariably progressing to diabetes. This is not associated with HLA haplotypes conferring susceptibility to or protection from IDDM.

Adolescent↗

PubMed1995

Radioimmunoassays for glutamic acid decarboxylase (GAD65) and GAD65 autoantibodies using 35S or 3H recombinant human ligands.

Autoantibodies are an important marker of human autoimmune diseases and the development of simple, precise and reproducible immunoassays to detect autoantibodies is important to our understanding of human autoimmunity. GAD65 autoantibodies occur frequently in insulin-dependent diabetic patients and is a useful marker for IDDM. A RIA to detect immunoreactive GAD65 has not been described. In the present study we describe a semi-automated fluid-phase immunoassay for the rapid detection of GAD65 autoantibodies in human serum. We also developed a sensitive RIA to determine immunoreactive human GAD65 in biological fluids and in vitro cell systems. Using in vitro translated recombinant human GAD65 in a multiwell-adapted procedure, our GAD65Ab RIA combines high specificity and sensitivity with a high capacity to analyze a large number of samples. In this report the three critical steps in the GAD65Ab RIA, DNA preparation, in vitro translation and immunoprecipitation, have been optimized. In our RIA, GAD65Ab were detected in 116/155 (75%) new onset Swedish IDDM children and in 1/85 (1.2%) healthy controls. In an immunoassay to detect autoantibodies against the proinsulin converting enzyme 2 (PC-2) no such antibodies were detected in IDDM patients. In the GAD65 RIA the lower detection limit was 2 ng/ml (31 fmol/ml). Our data demonstrate that autoantigen radioligands produced by in vitro translation are useful in RIA for autoantibodies and autoantigens in studies of human autoimmunity.

Adolescent↗

PubMed1995

Targeting of the 67-kDa isoform of glutamic acid decarboxylase to intracellular organelles is mediated by its interaction with the NH2-terminal region of the 65-kDa isoform of glutamic acid decarboxylase.

The two isoforms of glutamic acid decarboxylase (GAD), GAD67 and GAD65, synthesize the neurotransmitter gamma-aminobutyric acid in neurons and pancreatic beta-cells. Previous studies suggest that GAD67 is a soluble cytosolic protein, whereas GAD65 is membrane-associated. Here, we study the intracellular distribution of GAD67 in neurons, pancreatic beta-cells, and fibroblasts transfected either with GAD65 and GAD67 together or with GAD67 alone. Neuronal GAD67 is partially recovered with GAD65 in membrane-containing pellet fractions and Triton X-114 detergent phases. The two proteins co-immunoprecipitate from extracts of brain and GAD65-GAD67 co-transfected fibroblasts, but not when extracts of GAD65 and GAD67 transfected fibroblasts were mixed and used as a starting material for immunoprecipitation. GAD67 is concentrated in the Golgi complex region in GAD65-GAD67 co-transfected fibroblasts, but not in fibroblasts transfected with GAD67 alone. A pool of neuronal GAD67 co-localizes with GAD65 in the Golgi complex region and in many synapses. The two proteins also co-localize in the perinuclear region of some pancreatic beta-cells. GAD67 interacts with the NH2-terminal region of GAD65, even in the absence of palmitoylation of this region of GAD65. Taken together, our results indicate that GAD65-GAD67 association occurs in vivo and is required for the targeting of GAD67 to membranes.

Animals↗

PubMed1995

Flow injection fluorescence microscopy applied to a rapid cell surface immunoassay.

A perfusion system for fluorescence microscopy that utilized a flow injection system was developed and used to study cell surface antibody binding on viable cells grown in monolayer cultures on coverslips. A polyclonal cell-specific antiserum used to probe the cell surface was monitored by indirect immunofluorescence. The flow injection system was completely automatic and allowed controlled perfusion of cell surfaces with the desired sequence of antibodies. The individual steps of the indirect assay were studied to determine the binding behavior of the primary cell surface antibody, the labeled second antibody, and control of nonspecific binding. Under stopped flow conditions, the second antibody was maximally bound within 10 min, while constant mixing of the second antibody solution over the cells resulted in maximal binding within as little as 6 min. A primary antibody contact time of 2 min followed by a wash and then exposure to the second antibody for 2 min showed that this fast automated procedure could distinguish specific from nonspecific binding of cell surface antibodies to the same set of cells for several repeated exposures. The flow injection fluorescence microscopy system can be automated to allow screening for cell surface antibodies and to study their interaction with specific cell surface antigens.

Animals↗

PubMed1995

Heterogeneity of islet pathology in two infants with recent onset diabetes mellitus.

The mechanisms by which the beta cells of pancreatic islets are destroyed in insulin-dependent diabetes mellitus (IDDM) are poorly understood. In this report the pancreatic histo- and immunopathology of two children, both HLA-DR 3/4, DQ 2/8 positive and who both died from cerebral oedema within a day of clinical diagnosis of IDDM, were investigated. Patient 1, a 14-month-old girl, had a 4-week history of polydipsia and polyuria. Patient 2, a 3-year-old boy, had 2 days of illness. Both patients had a similarly severe loss of insulin cells but differed markedly as to the extent of lymphocytic islet infiltration (insulitis). Apart from insulitis, marked islet macrophage infiltration was demonstrated in both patients with the HAM-56 monoclonal antibody. Neither patient showed aberrant expression of HLA class II antigens on insulin-immunoreactive cells, but allele-specific HLA-DQ8 expression was evident on endothelial cells. Glutamic acid decarboxylase immunoreactivity was detected in both insulin- and glucagon-immunoreactive cells. It is concluded that the heterogeneity of islet pathology, especially insulitis, may reflect different dynamics and extent rather than different pathomechanisms of immune destruction of islets in IDDM.

Child, Preschool↗

PubMed1995

Predictability of heterozygosity scores and polymorphism information content values for rat genetic markers.

Construction of a genetic linkage map of the laboratory rat, Rattus norvegicus, establishes the rat as a genetic model. Allele sizes were reported for 432 simple sequence length polymorphisms (SSLPs) genotyped in 12 different substrains belonging to nine different inbred strains of rats. However, these nine strains represent only a fraction of the more than 140 inbred strains available. If allele sizes are not known, alternative indices of markers' polymorphism content can be used, such as heterozygosity (H) and polymorphism information content (PIC). Here, we have determined heterozygosity scores and PIC values for all markers of the rat genetic linkage map, and we evaluate the predictability of the heterozygosity and the PIC values. Correlation analysis between the nine inbred strains reported for the rat map and ten "test" strains yielded r = 0.42 and r = 0.44 for heterozygosity and PIC values, respectively. While the correlation of the indices between the two groups of animals is low, these indices do provide a means of predicting whether a genetic marker will be informative in strains where allele sizes are not known.

Animals↗

PubMed1995

Sexual dimorphism in transmission of expression of islet autoantibodies to offspring.

To help elucidate the mode of inheritance of insulin-dependent diabetes mellitus (IDDM), we measured GAD (glutamic acid decarboxylase) autoantibodies (GAD65Ab), insulin autoantibodies (IAA), and cytoplasmic islet cell autoantibodies (ICA) in 292 sequentially screened non-diabetic offspring of patients with IDDM. The prevalence of these islet autoantibodies was higher in offspring of diabetic fathers than in offspring of diabetic mothers. The prevalences of GAD65Ab, IAA, and ICA in the offspring of diabetic fathers were 11.5%, 10.8%, and 8.1% vs 2.1%, 1.4%, and 2.8%, respectively in the offspring of diabetic mothers (p < 0.002, p < 0.001, and p = 0.06 NS). Amongst autoantibody-positive relatives the IAA and ICA levels were significantly higher in offspring of diabetic fathers than of diabetic mothers (p < 0.002 and p < 0.01, respectively). The frequencies of these autoantibodies were equal in male and female offspring. We conclude that IDDM mothers transmitted islet autoimmunity less frequently to their offspring than IDDM fathers. Given the markedly lower frequency of autoantibodies in offspring of mothers, larger sample sizes will be required to determine whether islet autoantibodies are influenced by age of IDDM onset of mothers, maternal age of pregnancy, and presence of diabetes in these mothers prior to conception.

Adolescent↗

PubMed1995

Glutamate decarboxylase antibody levels predict rate of beta-cell decline in adult-onset diabetes.

Glutamate decarboxylase autoantibodies (GAD65Ab) and beta-cell function were evaluated at and 3 years after diabetes onset in consecutive subjects over 15 years of age. At onset, 21/32 (66%) insulin-treated patients (mean age 43, range 16-79 years) had GAD65Ab; all GAD65Ab persisted 3 years later. At onset, 20/82 (24%) non-insulin-treated patients (mean age 56, range 20-79 years) had GAD65Ab. Of those with persistent GAD65Ab, 8 non-insulin-treated and 11 insulin-treated patients consented to follow-up glucose and glucagon stimulation tests. For non-insulin-treated patients, quantitative GAD65Ab index at onset correlated inversely with 1 + 3 min C-peptide response to glucose (r = -0.68, P < 0.05) and to glucagon (r = -0.79, P < 0.05) 3 years later. Those with high (> 0.50) initial GAD65Ab index had lower C-peptide (fasting, 1 + 3 min after glucose and after glucagon) 3 years later, versus those with low (< 0.50) initial GAD65Ab index (P < 0.05). In conclusion, not only did GAD65Ab presence predict future insulin dependence, but higher GAD65Ab levels may mark more rapid decline in beta-cell function in apparent non-insulin-dependent diabetes.

Adolescent↗

PubMed1995

Thyroiditis in the BB rat is associated with lymphopenia but occurs independently of diabetes.

The spontaneously diabetic BB rat is an excellent and well studied model for human insulin-dependent diabetes (IDDM), sharing many important features with the human disease. Similarities include an equal frequency of IDDM in males and females, production of antibodies against pancreatic cell antigens, and an MHC disease association. In addition, the BB rat shares with human IDDM patients an increased frequency of autoantibodies against the parietal cells of the stomach and colloid cells of the thyroid gland. Here we investigate the genetic basis of thyroiditis in the BB rat. Based on crosses between BB, Lewis and Fischer rats, we show that two susceptibility factors for diabetes--the lymphopenia trait present in diabetes prone BB rats and the MHC--also appear to be risk factors for thyroiditis. However, the nature of the susceptibility was different for the two autoimmune diseases, with lymphopenia being absolutely required for diabetes although it only conferred increased risk for thyroiditis. Also, in contrast to IDDM, the MHC conferred dominant susceptibility to thyroiditis. Despite these shared risk factors, diabetes per se did not show significant correlation with thyroiditis.

Animals↗

PubMed1995

Pathogenesis of insulin-dependent diabetes mellitus.

Insulin-dependent diabetes mellitus is strongly associated with certain HLA types and the presence of islet cell-specific autoantibodies. The pathogenesis is a specific loss of pancreatic beta cells. The dissection of IDDM genes is complicated by the low recurrence rate of the disease among first-degree relatives. HLA-DQ2 and 8 are closest to IDDM with a marked synergistic effect of DQ2/8 heterozygotes. The associations with other HLA genes are often explained by linkage disequilibrium. Genetic factors on other chromosomes which influence the pathogenesis are still to be fully identified but candidates are on chromosomes 11 (insulin gene polymorphisms) and 7 (TCR gene polymorphisms). The autoreactivity against the GAD65 isoform is pronounced both before and at the clinical onset of IDDM. GAD65 autoantibodies show the highest predictive value and may represent an initiating autoantigen. Autoantibodies to numerous other beta cell autoantigens are detected at the clinical onset but may represent a secondary response and antigen spreading during a sustained autoimmune attack on the beta cells. The role of T cells in human IDDM is yet to be defined. GAD65 and other islet autoantibodies have a low positive predictive value for IDDM and further investigations are needed to clarify ways to predict IDDM in the general population.

Animals↗

PubMed1995

A genetic linkage map of the laboratory rat, Rattus norvegicus.

We report the construction of the first complete genetic linkage map of the laboratory rat. By testing 1171 simple sequence length polymorphisms (SSLPs), we have identified 432 markers that show polymorphisms between the SHR and BN rat strains and mapped them in a single (SHR x BN) F2 intercross. The loci define 21 large linkage groups corresponding to the 21 rat chromosomes, together with a pair of nearby markers on chromosome 9 that are not linked to the rest of the map. Because 99.5% of the markers fall into one of the 21 large linkage groups, the maps appear to cover the vast majority of the rat genome. The availability of the map should facilitate whole genome scans for genes underlying qualitative and quantitative traits relevant to mammalian physiology and pathobiology.

Animals↗

PubMed1995

Effects of the second HLA-DQ haplotype on the association with childhood insulin-dependent diabetes mellitus.

Analysis of HLA-DQ molecules in a large study comprising 425 consecutively diagnosed Swedish Caucasians with IDDM and 367 age, sex and geographically matched controls confirms previous observations that: (a) DQB1*0302-DQA1*0301 confer susceptibility in a dominant manner, except when they are associated with DQB1*0602-DQA1*0102; (b) DQB1*0501-DQA1*0201 does not confer susceptibility to IDDM in either homozygous or heterozygous combinations with any other DQ molecules except when it is in association with DQB1*0302-DQA1*0301; (c) heterozygous combinations of DQB1*0302-DQA1*0301 and DQB1*0501-DQA1*0201 confer the highest risk to IDDM; (d) in DQ2 positive patients being negative for DQ8 in second haplotype (n = 58) the susceptibility may be explained by DR, since all these patients were DR3 positive, or by unknown factors between DQ and DR and (e) DQB1*0602-DQA1*0102 confers protection in a dominant manner. This large study does not confirm the positive association previously observed in Norwegians between the DQ8/DQ4 genotype and IDDM, as this genotype was not significantly associated with IDDM in Swedish patients. The new findings in this study include (a) that DQ8/DQ6 (DQB1*0604-DQA1*0102) was associated with IDDM in Swedish patients and (b) analysis of individual amino acids in DQB1 chain does not fully explain susceptibility to IDDM.

Adolescent↗

PubMed1995

Population analysis of protection by HLA-DR and DQ genes from insulin-dependent diabetes mellitus in Swedish children with insulin-dependent diabetes and controls.

A negative association between insulin-dependent diabetes mellitus (IDDM) and HLA-DR, DQA1 or DQB1 was found in a large population-based investigation of childhood-onset patients (more than 420 patients) and controls (more than 340 controls) from Sweden. The relative risk was decreased for several haplotypes that were negatively associated with IDDM: DR15-DQA1*0102-DQB1*0602, DR7-DQA1*0201-DQB1*0303, DR14-DQA1*0101-DQB1*0503, DR11-DQA1*0501-DQB1*0301, DR13-DQA1*0103-DQB1*0603 and DR4-DQA1*0301-DQB1*0301. In a relative predispositional effect (RPE) analysis, however, only the DR15-DQA1*0102-DQB1*0602 haplotype was significantly decreased, which suggests that the major protective effect for IDDM is carried by this haplotype. This was supported by the observation that all genotypes which were negatively associated with IDDM, except DR7/13, included at least one allele from the DR15-DQA1*0102-DQB1*0602 haplotype. Relative predispositional effect (RPE) analysis of genotypes showed further that the DR15-DQA1*0102-DQB1*0602 haplotype was also negatively associated with IDDM when combined with any other haplotype, whether negatively or positively associated with IDDM. This supports previous suggestions that DR15-DQA1*0102-DQB1*0602 acts dominantly. However, both the stratification and the predispositional allele test failed to distinguish the negative association between IDDM and DR15 from that of DQB1*0602. On the other hand, these tests indicated that DQA1*0102 was not likely to explain the negative association between IDDM and the DR15-DQA1*0102-DQB1*0602 haplotype. We conclude that the major protective effect for IDDM in the population of Swedish children is conferred by the DR15-DQA1*0102-DQB1*0602 haplotype in a dominant fashion, the DQB1*0602 allele being the allele most likely to be responsible for the protective effect of this haplotype, although an effect of the DR15 allele could not be excluded.

Adolescent↗

PubMed1995

Glutamate decarboxylase-, insulin-, and islet cell-antibodies and HLA typing to detect diabetes in a general population-based study of Swedish children.

Most autoimmune diabetes occurs in those without a diabetic relative, but few cases are identifiable prospectively. To model general population prediction, 491 consecutive newly diabetic children from all of Sweden were tested for autoantibodies to glutamate decarboxylase (GAD65ab), insulin (IAA), and islet cells (ICA), and for HLA-DQ genotypes by PCR; 415 matched control children were tested in parallel. GAD65ab sensitivity/specificity was 70/96%, versus 84/96% for ICA, 56/97% for IAA, 93/93% (any positive), 39/99.7% (all positive), and 41/99.7% (GAD65ab plus IAA). The latter's 25% predictive value was not improved by requiring concomitant high-risk HLA genotypes. GAD65ab were associated with DQA1*0501/B1*0201 (DQ2; P = 0.007) but not DQA1*0301/B1*0302 (DQ8), and IAA with DQA1*0301/B1*0302 (DQ8; P = 0.03) but not DQA1*0501/B1*0201 (DQ2). GAD65ab were more prevalent in females than males (79 vs. 63%; P < 0.0001) but did not vary with onset age nor season. Combining the three antibody assays yielded sufficient sensitivity for screening. GADab were relatively sensitive/specific for diabetes, but even with HLA marker combinations yielded predictive values insufficient for early immunointervention in the low-prevalence general population.

Adolescent↗

PubMed1995

Differential detection of rat islet and brain glutamic acid decarboxylase (GAD) isoforms with sequence-specific peptide antibodies.

We studied the distribution of the M(r) 65,000 and M(r) 67,000 isoforms of glutamic acid decarboxylase, GAD65 and GAD67, in rat islets and brain by immunocytochemistry. Synthetic peptides representing selected GAD65 or GAD67 sequences were used to produce sequence-specific antibodies, allowing differential immunocytochemical detection of the two isoforms. GAD-specific reactivity of each peptide antiserum was confirmed by ELISA, immunoblotting, and immunoprecipitation. Immunostaining specificity was verified by displacement with either immunizing or irrelevant peptide. Dual immunostaining with GAD isoform-specific antibodies and polyclonal antibodies to glucagon showed that GAD65 was primarily detected in rat pancreatic islet beta-cells, whereas alpha-cells had weak GAD65 staining. In contrast, GAD67 was detected primarily in alpha-cells. In rat brain, GAD65 and GAD67 were present in neuron cell bodies and processes. These data demonstrate that antibodies raised against the N-terminus of GAD allow differential immunocytochemical identification of GAD67 and GAD65. Differential expression of GAD isoforms within islet alpha- and beta-cells supports the role of GAD65 in autoimmune diabetes and stiff-man syndrome.

Animals↗

PubMed1995

High diagnostic sensitivity of glutamate decarboxylase autoantibodies in insulin-dependent diabetes mellitus with clinical onset between age 20 and 40 years. The Belgian Diabetes Registry.

Patients with adult-onset Type 1 (insulin-dependent) diabetes mellitus (IDDM) are more difficult to identify than young patients, as their clinical onset is often less acute with a questionable state of insulin dependency. Classification may be facilitated by the detection of autoantibodies that are associated with IDDM. The prevalence of islet cell autoantibodies (ICA) and insulin autoantibodies (IAA) is, however, markedly lower in adult than in young patients. The present study assesses the usefulness of antibodies against glutamate decarboxylase (GAD), as a complementary marker. Sera from 312 recent-onset IDDM patients under age 40 and 163 age-matched controls were assayed for IAA, ICA, and antibodies against recombinant GAD65 (M(r) 65,000) or GAD67 (M(r) 67,000). IAA or ICA occurred in over 90% of patients diagnosed under age 20 but only in 65% of patients between age 20 and 40. Determination of GAD65-Ab did not increase the percent antibody positive patients under age 10, but did so at older ages: from 92-98% in the 10-19 years age group, and from 65-85% in the 20-39 years age group. The determination of GAD67-Ab did not add to the information provided by the GAD65-Ab assay. Our results indicate that, alone or in combination with ICA, the GAD65-Ab assay identified more patients with an IDDM marker in the age group 20-39 years than in the group under age 20.

Adolescent↗

PubMed1995

High diagnostic accuracy for idiopathic Addison's disease with a sensitive radiobinding assay for autoantibodies against recombinant human 21-hydroxylase.

Autoantibodies against 21-hydroxylase (P450c21) are common in idiopathic autoimmune Addison's disease. In the present work, we have developed a sensitive radiobinding assay using in vitro translated recombinant human 35S-P450c21. Levels of P450c21 antibodies (P450c21-Ab) were expressed as a relative index (P450c21 index) using a P450c21-Ab positive Addisonian serum and two antibody-negative healthy sera as positive and negative standards in healthy individuals. The upper level of normal was the mean + 3 SD. Positivity for P450c21-Ab was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of immunoprecipitated 35S-P450c21. In 38 Addisonian patients, P450c21-Ab were found in 24/28 (86%) idiopathic, 0/5 post-tuberculosis, 0/3 adrenoleukodystrophy, and 0/2 post-adrenalectomy sera. Among healthy individuals, 1/70 (1.4%) were positive. The P450c21 index, as an estimate of P450c21-Ab levels, correlated inversely with the duration of idiopathic Addison's disease (r = -0.527; P = 0.007): 16/16 (100%) positive in patients with less than 20 yr and 8/12 (67%) positive in patients with more than 20 yr disease duration. The availability of this simple and sensitive radiobinding assay to evaluate levels of P450c21-Ab will permit large clinical studies as well as screening subjects at risk. In addition, the general population can now be screened to evaluate the predictive value of P450c21-Ab for Addison's disease.

Acute Disease↗