Biomedical subjects
D Schatz
Publications and source records attributed to D Schatz.
Pharmacological approaches to the prevention of autoimmune diabetes.
Insulin-dependent (type I) diabetes mellitus (IDDM) is the consequence of a chronic cell-mediated immune attack upon the insulin-producing beta-cells. Progressive insulinopenia is characteristic of individuals who eventually develop IDDM. Autoimmunity develops because of a failure in self-nonself discrimination. Autoimmunity is usually detected when autoantibodies are present in the patient's serum. However, autoantibodies are not synonymous with disease, as many autoantibody-positive individuals show no evidence of clinical disease. Studies initiated in the early 1980s demonstrated that short term remission from IDDM could be induced or lengthened with immunosuppressive therapy. However, no long term remissions were achieved. Current prevention strategies use a combination of autoantibody marker testing and beta-cell function testing to identify individuals with 'prediabetes'. The most useful autoantibodies for prediabetes screening include islet cell autoantibodies, insulin autoantibodies, glutamic acid decarboxylase autoantibodies and IA-2 autoantibodies. Immunointervention techniques have focused on protecting beta-cells from oxidative damage and developing tolerance to beta-cell autoantigens. Environmental manipulation may also be of benefit but its effectiveness is unproven. The pharmacist of the future may be involved in dispensing autoantigens, cytokines, anti-cytokine antibodies, anti-cytokine receptor antibodies, vaccines or viral vectors for gene therapy in the prevention of IDDM.
Autoantibodies to the extracellular domain of the calcium sensing receptor in patients with acquired hypoparathyroidism.
Acquired hypoparathyroidism (AH) has been considered to result from an autoimmune process but the self-antigens have not been identified. We studied 25 patients with AH, of which 17 had type I autoimmune polyglandular syndrome and 8 had AH associated with autoimmune hypothyroidism. Five of 25 (20%) AH sera reacted to a membrane-associated antigen of 120-140 kD in human parathyroid gland extracts using immunoblot analysis. This is the exact size of the calcium sensing receptor (Ca-SR). The AH sera were then tested by immunoblot using a membrane fraction of HEK-293 cells transfected with Ca-SR cDNA. Eight of 25 (32%) AH sera reacted to a 120-140-kD protein, which closely matched that recognized by the anti-Ca-SR IgG raised in rabbits. The Ca-SR cDNA was translated in vitro into two parts in order to identify the antigenic epitopes. By using this technique, 14 of 25 (56%) AH sera were positive to the extracellular domain of the Ca-SR, whereas none of the AH patients sera reacted to the intracellular domain. The reactivity of the positive sera was completely removed after pre-absorption with the Ca-SR containing membranes. Sera from 50 patients with various other autoimmune diseases as well as 22 normal controls were also tested, and none of them was positive. In conclusion, the Ca-SR has been identified as an autoantigen in AH.
Although DR3-DQB1*0201 may be associated with multiple component diseases of the autoimmune polyglandular syndromes, the human leukocyte antigen DR4-DQB1*0302 haplotype is implicated only in beta-cell autoimmunity.
Human leukocyte antigen (HLA)-DRB1 and -DQB1 alleles were analyzed using a PCR-based sequence-specific priming technique in 16 patients with autoimmune polyglandular syndrome type I (APS-I), 31 patients with APS-II, and 110 patients with component diseases of APS-II, including 9 patients with isolated Addison's disease, 43 patients with Hashimoto's thyroiditis, 22 patients with Graves' disease, and 36 patients with vitiligo. No significant associations was observed between HLA and APS-I patients in our data set, nor was sharing of HLA haplotypes by sibling pairs affected by APS I significantly different from the random expectation. Thus, HLA-DRB1 and -DQB1 genes are probably not involved in APS-I. To delineate the associations between HLA-DRB1, DQB1, and APS-II, we analyzed APS-II patients with or without beta-cell autoimmunity [i.e. insulin-dependent diabetes (IDD) and/or islet cell or glutamic acid decarboxylase autoantibodies]. Our results suggest that the association between DR4-DQB1*0302 and APS-II was entirely due to the presence of pancreatic beta-cell autoimmunity, since this haplotype was otherwise not significantly associated with APS-II or with any other of its component diseases. In contrast, the DR3-DQB1*0201 haplotype was associated not only with IDD, but also with APS-II in the absence of pancreatic beta-cell autoimmunity, as were several its component diseases, including isolated Addison's disease, Graves' disease, and Hashimoto's thyroiditis. Interestingly, the frequency of DQB1*0602, a dominantly protective allele against IDD, was not significantly decreased in the APS-II patients with IDD or beta-cell autoimmunity, albeit the patient numbers were small. This phenomenon may suggest that the development of autoimmunity to nonpancreatic endocrine glands may predispose autoimmunity to the pancreatic beta-cells and involve genes other than those of the MHC.
Recent advances in the immunopathogenesis of insulin-dependent diabetes mellitus.
Insulin-dependent diabetes mellitus places a major burden on both affected individuals and society because of its high morbidity and mortality rates and financial costs. That the disease occurs in genetically susceptible individuals as a result of an immunologically mediated process thought to be triggered by environmental factors probably operating in early childhood is well established. Enhanced knowledge of the immunopathogenesis, genetics, and natural history of insulin-dependent diabetes mellitus in nonobese diabetic mice and humans has enabled investigators to better predict disease onset and design therapies aimed at its prevention. Major national and international multicenter trials are currently in progress, engendering cautious optimism that the disease may safely be prevented.
GAD65 autoantibodies increase the predictability but not the sensitivity of islet cell and insulin autoantibodies for developing insulin dependent diabetes mellitus.
The clinical onset of insulin-dependent diabetes (IDD) can be predicted by determination of autoantibodies to several pancreatic-islet cell antigens. Islet-cell autoantibodies (ICA) and insulin autoantibodies (AA) are most commonly used. We have developed a recombinant human glutamic acid decarboxylase (GAD65) radioimmunoassay and measured autoantibodies to GAD65 (GAD65A) in the sera of 73 documented prediabetic individuals, 76 newly-diagnosed patients, 103 relatives of IDD probands at increased risk for the development of IDD because they were positive for ICA and/or IAA, 72 ICA and IAA negative relatives, and 207 healthy controls. Our data demonstrate that GAD65A are strongly associated with the currently established autoantibody markers of IDD. Their presence in prediabetic subjects with only ICA or IAA enhances their risk for progression to IDD, yet does not much enhance the screening sensitivity already available through conventional ICA and IAA for IDD prediction.
High risk HLA-DR/DQ genotypes for IDD confer susceptibility to autoantibodies but DQB1*0602 does not prevent them.
Although HLA class II genes are important in insulin-dependent diabetes (IDD), their influence on the expression of IDD-associated autoantibodies (aAb) is unclear. We compared HLA-DRB1 and DQB1 gene frequencies in several Caucasian groups: 191 normal controls, 378 IDD patients, and 357 non-diabetic relatives of which 250 had no aAb, 107 had at least one aAb (79 ICA+, 31 GAD65+ and 49 IAA+), and 23 had both ICA+ and IAA+. We found that the frequencies of DR3/4 or DQB1*0201/0302 heterozygotes were significantly higher in aAb+ relatives compared to aAb- relatives. The frequencies of DR4/4 or DR4/X (X = non 3 or 4) and DQB1*0302/X (X = 0201 or 0302) in aAb+ relatives were not different from the aAb- relatives (which were enriched for these haplotypes), but were significantly higher than normal controls. The frequencies of DR3/X or DQB1*0201/X were decreased in both aAb+ relatives and IDD patients. Interestingly, the dominant IDD-protective DQB1*0602 allele allowed the development of individual aAbs (10% of ICA+ and 8% IAA+ relatives had the allele), but was not observed in any high risk double aAb+, or GAD65Ab+ relatives. The latter finding was similar to that in our patients with IDD, in that only two of them (0.5%) had a DQB1*0602 allele. In conclusion, HLA-encoded susceptibilities to disease-relevant autoantibody production and IDD are concordant with the susceptibility alleles, but discordant for the protective DQB1*0602. Thus HLA genotyping for DQB1*0602 would impact on the selection of aAb+ relatives for disease prevention trials.
Islet cell antibodies predict insulin-dependent diabetes in United States school age children as powerfully as in unaffected relatives.
Islet cell antibodies (ICA) in the sera of nondiabetic relatives of patients with insulin-dependent diabetes (IDD) are predictive of the disease, a finding that permits the design of intervention strategies to prevent it. However, 85% or more of patients with new onset IDD have no affected relative. We therefore screened 9,696 schoolchildren between the ages of 5 and 18 yr (mean age 10.7 yr) in Pasco County, Florida for ICA in three surveys during 1984/5, 1987/8, and 1990/1 and have followed them prospectively. Approximately 4,000 of these children have been followed for nearly 8 yr. ICA titers > or = 10 Juvenile Diabetes Foundation units on replicate tests were detected in 57 of the children (0.59%). 10 children have developed diabetes so far, and all had ICA detected beforehand. The likelihood of developing IDD among the ICA-positive children was compared with 2,959 age-matched nondiabetic first degree relatives of IDD probands who were screened for ICA by our laboratory during the same time period and also followed prospectively. Of 103 (3.5%) ICA-positive relatives, 31 have developed IDD. Life table analysis reveals no statistically significant differences in the probability of developing IDD between the ICA-positive schoolchildren and ICA-positive first degree relatives (P = 0.3). The estimated risk of developing IDD by 7 yr in the ICA-positive schoolchildren was 45% (95% confidence interval 15-74%) compared with 43% (confidence interval 22-63%) in the relatives. We conclude that ICA appear to be as predictive of IDD in low-risk schoolchildren as they are in high-risk relatives. These data suggest that it is feasible to predict IDD by screening a general population of schoolchildren for ICA and that those found to be positive could be considered, in addition to relatives, for intervention protocols to prevent the disease.
Glycemic control in a sample of black and white clinic patients with NIDDM.
OBJECTIVE: To compare glycemic control of black and white patients under treatment for non-insulin-dependent diabetes mellitus (NIDDM). RESEARCH DESIGN AND METHODS: Medical records of patients with NIDDM were reviewed at 19 of 24 clinics or health centers in a specified area of north central Florida. Data were abstracted from the charts of all non-Hispanic black (n = 248) or white (n = 280) patients who met preset eligibility criteria. RESULTS: The mean +/- SD age was 58 +/- 14 years; the mean +/- SD duration of diabetes was 9 +/- 7.5 years. Of 528 patients, 220 were treated with oral hypoglycemic agents, 269 were treated with insulin, 28 were treated with diet alone, and 11 were treated with a combination of oral agents, diet, and insulin. Glycosylated hemoglobin was > or = 8% for 47% of black women, 41% of black men, 38% of white men, and 29% of white women. The less favorable control status of the black women persisted within categories of age (40-59 years or > or = 60 years), treatment (insulin or oral hypoglycemic agents), time since diagnosis (less than versus greater than or equal to the median of 7 years), and obesity (noted versus not noted in chart). In multiple logistic regression analyses controlling simultaneously for these variables, the odds (95% confidence interval [CI]), compared with white women, of having glycosylated hemoglobin > or = 8% were 2.2 (1.4, 3.4) for black women and 1.5 (0.8, 2.9) and 1.4 (0.8, 2.5) for black and white men, respectively. CONCLUSIONS: Black women were more likely than white women or men to have glycosylated hemoglobin > or = 8%. This difference was not readily explained by age, type of treatment, time since diagnosis, or a notation of obesity in the medical record, although an effect of obesity cannot be excluded on this basis.
Early diagnosis and specific treatment of insulin-dependent diabetes.
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The genetics of autoimmune diabetes. Approaching a solution to the problem.
Molecular genetics has had a substantial impact on our understanding of inherited susceptibility to insulin-dependent diabetes mellitus. Alleles at the HLA-DQA1 and DQB1 loci appear to have the greatest influence on diabetogenesis. Other promising loci are present on chromosome 11 in the vicinity of the insulin gene. We have sought not only to improve our prediction of insulin-dependent diabetes mellitus but also to reveal the underlying immune and nonimmune defects that predispose to autoimmune beta-cell destruction. Continued advances in the field of genetics will aid in the prevention of insulin-dependent diabetes mellitus, which is our ultimate goal.
Antigen-specific immunotherapy: oral tolerance and subcutaneous immunization in the treatment of insulin-dependent diabetes.
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Insulin and islet cell autoantibodies as time-dependent covariates in the development of insulin-dependent diabetes: a prospective study in relatives.
Using time-dependent methods, the temporal relationships between the detection of insulin and islet cell autoantibodies and the onset of insulin dependent diabetes (IDDM) were analyzed in a prospective study of 4694 nondiabetic relatives of 1929 patients with IDDM who had been followed for a median of 4 yr. Insulin autoantibodies were detected in 1.5% of relatives at their initial test whereas an additional 1.0% subsequently became positive for these antibodies during follow-up. Islet cell autoantibodies were detected in 2.6% of the relatives at the time of their first test and an additional 0.9% were observed to develop them during the follow-up period. The risk of developing IDDM was significantly higher (P = 0.0001) among those who were found to have one of these antibodies, but was highest among those under the age of 20 yr at inception of this study who tested positive for both. Among older relatives, the detection of insulin autoantibodies among those who were islet cell antibody positive did not convey an additional risk of IDDM. In a subset of relatives, the presence of either antibody was associated with a higher frequency (P < 0.001) of diabetes associated human leukocyte antigen-DR 3/4 heterozygotes. Islet cell autoantibodies were highly associated with elevated fasting and 60-min glucose concentrations (P = 0.0001) as well as decreased early phase (1 and 3 min) insulin response to an iv glucose tolerance test (P = 0.0001). Insulin antibodies were significantly associated with decreased early phase insulin response to iv glucose (P = 0.0003). These data confirm independent risks associated with each antibody and suggest that their temporal relationship may be an important reflection of the pathogenic process underlying IDDM observations which facilitate its predictability.
Determinant nucleotides of yeast tRNA(Asp) interact directly with aspartyl-tRNA synthetase.
The interaction of wild-type and mutant yeast tRNA(Asp) transcripts with yeast aspartyl-tRNA synthetase (AspRS; EC 6.1.1.12) has been probed by using iodine cleavage of phosphorothioate-substituted transcripts. AspRS protects phosphates in the anticodon (G34, U35), D-stem (U25), and acceptor end (G73) that correspond to determinant nucleotides for aspartylation. This protection, as well as that in anticodon stem (C29, U40, G41) and D-stem (U11 to U13), is consistent with direct interaction of AspRS at these phosphates. Other protection, in the variable loop (G45), D-loop (G18, G19), and T-stem and loop (G53, U54, U55), as well as enhanced reactivity at G37, may result from conformational changes of the transcript upon binding to AspRS. Transcripts mutated at determinant positions showed a loss of phosphate protection in the region of the mutation while maintaining the global protection pattern. The ensemble of results suggests that aspartylation specificity arises from both protein-base and protein-phosphate contacts and that different regions of tRNA(Asp) interact independently with AspRS. A mutant transcript of yeast tRNA(Phe) that contains the set of identity nucleotides for specific aspartylation gave a phosphate protection pattern strikingly similar to that of wild-type tRNA(Asp). This confirms that a small number of nucleotides within a different tRNA sequence context can direct specific interaction with synthetase.
Interaction of Escherichia coli tRNA(Ser) with its cognate aminoacyl-tRNA synthetase as determined by footprinting with phosphorothioate-containing tRNA transcripts.
A footprinting technique using phosphorothioate-containing RNA transcripts has been developed and applied to identify contacts between Escherichia coli tRNA(Ser) and its cognate aminoacyl-tRNA synthetase. The cloned gene for the tRNA was transcribed in four reactions in which a different NTP was complemented by 5% of the corresponding nucleoside 5'-O-(1-thiotriphosphate). The phosphorothioate groups of such transcripts are cleaved by reaction with iodine to permit sequencing of the transcripts. Footprinting was achieved by performing the same reaction with the phosphorothioate-tRNA-enzyme complex. At 1 mM iodine, selective protection of the tRNA transcripts in the cognate system was observed, with strong protection at positions 52 and 68 and weak protection at positions 46, 53, 67, 69, and 70. It is suggested that these regions of the tRNA interact with the helical arm of the synthetase.
The parental origin of the single X chromosome in Turner syndrome: lack of correlation with parental age or clinical phenotype.
We have used X- and Y-linked RFLPs to determine the origin of the single X chromosome in 25 live-born individuals with Turner syndrome. We determined that 18 individuals retained a maternal X (Xm) and that seven retained the paternal X (Xp). No occult mosaicism was detected. We found no differences in either maternal or paternal ages for the two groups. The ratio of maternal X to paternal X is just over 2:1, which is consistent with the expected proportion of meiotic or mitotic products, with equal loss at each step, given the nonviability of 45,Y. Six phenotypic or physiologic characteristics were assessed: (1) birth weight, (2) height percentile at time of testing, (3) presence of a webbed neck, (4) cardiovascular abnormalities, (5) renal abnormalities, and (6) thyroid autoimmunity. There were no significant differences in birth weights or heights between the girls who retained the maternal X or the paternal X. In addition, no differences between the groups could be appreciated in the incidence of the physical, anatomic, or physiologic parameters assessed.
Initial pathogenic events in IDDM.
A workshop sponsored by the National Institute of Child Health and Human Development was held in June 1988 to discuss the initial events in the pathogenesis of insulin-dependent diabetes and to make recommendations for future studies. Better definition of immunological markers to reliably predict the disease will enable the detection and study of the earliest pathogenic events involved. The precise autoimmune mechanisms and the role of the environment, both in the initiation of the disease process and precipitation of clinically overt disease, need to be accurately determined to define strategies that might eventually lead to its prevention.
The future of the imaging technologist.
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