Insulin-dependent diabetes: a disease of autoaggression.

Lymphocytes from patients with insulin-dependent diabetes demonstrated significant cytoadherence and cytotoxicity against human insulinoma cells in vitro as compared to lymphocytes from normals. Complement was not involved in insulinoma cell destruction. The findings suggest that insulin-dependent diabetes may be a disease of autoaggression.

Deficient Ganglioside Biosynthesis: a novel human sphingolipidosis.

An unusual lipid storage disese is chracterized by the accumulation of hematoside (Gms3) in the patient's liver and brain. In contrast to the other sphingoliidoses, the accumulation of Gm3 is not the result of a defective catabolic reaction, but is the first disorder caused by deficiency in ganglioside biosynthesis to be described in man.

Islet cell autoantigens in insulin-dependent diabetes.

A burgeoning number of antigenic targets of the islet cell autoimmunity in IDD have been identified, and more can be anticipated through improved methods for their identification. The challenge for those investigating the pathogenesis of IDD will be to assign the relative importance of these antigens to the development of the disease, and to resolve whether there is a dominant primary immunologic event that is followed by a series of secondary immunizations to a variety of normally sequestered islet cell antigens in the sequence of pathogenic events that culminate in IDD. One interesting observation that may have potential pathogenic implications is the observation that of all islet cell autoantigens described, only two (i.e., 64 kD/GAD, 38 kD) are reactive in their native configurations, implying that recognition of conformational epitopes is most important. This property argues for primary immunizing agents rather than secondary ones after release of denatured antigens and antigenic recognition through their epitopes. Given the complex and multiple physiological functions of islet cells and the continuous variation in their activity, it is reasonable to speculate that the speed of the progression to IDD could vary between individuals with respect to their insulin needs and the relative activities of their islets. Activated islets may express autoantigens that have only limited expression in quiescent islets. The often times striking variation in the severity of insulitis seen in different islets of a single pancreas may be explained by the level of activity of individual islets. Furthermore, disparity in HLA-DR/DQ associations with disease may involve differences in the immunological recognition of autoantigens. Whereas there is still much to learn, it is clear that disease predictability and disease intervention studies have been enhanced through the identification of the islet cell autoantigens in IDD.

Association of class II human histocompatibility leukocyte antigens with rheumatic fever.

The association of class I and II HLA antigens with rheumatic fever and its manifestations was examined in 72 patients, including 48 blacks and 24 Caucasians. No significant association was found between class I antigens and rheumatic fever. In contrast, HLA-DR2 and HLA-DR4 phenotypes were encountered in a significantly higher frequency in black and Caucasian patients with rheumatic fever, respectively, compared with the control populations (P less than 0.005). The most significant association (P less than 0.005) of these DR antigens with a major manifestation of rheumatic fever was found for mitral insufficiency. In addition, a significant association was encountered between persistent elevation of antibody to the group A streptococcal carbohydrate and HLA-DR4 in Caucasian patients (P less than 0.04) or HLA-DR2 in the black patients (P less than 0.001). The frequency of HLA-DR2/4 heterozygotes among patients with rheumatic fever did not differ significantly from controls. These findings support the concept of a genetically determined susceptibility to rheumatic fever and, particularly, to rheumatic heart disease. The association of the clinical manifestations of rheumatic fever and the immune hyperresponsiveness to a streptococcal antigen could be ascribed to a disease-associated immune-response gene which is in linkage disequilibrium with the DR2 and DR4 alleles of HLA-DR locus on chromosome six.

No evidence for serological autoimmunity to islet cell heat shock proteins in insulin dependent diabetes.

Recent studies in nonobese diabetic mice have implicated the autoimmune destruction of pancreatic islet cells with immunity to a beta cell protein cross-reactive to Mycobacterium tuberculosis heat shock protein 65 (hsp 65). Therefore, our studies examined serological immunity to islet cell hsp in humans with insulin-dependent diabetes (IDD). Heat shock of human islet cells in vitro markedly increased the synthesis of proteins of 72,000, 75,000, and 90,000 Mr. No autoantibodies reactive to these hsp, nor to the constituently expressed islet cell hsp 65 protein (identified as 60,000 Mr) were observed in IDD patients. The islet cell 64,000-Mr autoantigen and hsp 65 proteins were physiologically and immunocompetitively distinct. These experiments do not support the hypothesis that IDD in humans is associated with autoimmunity to islet cell heat shock proteins.

Cellular immunity to a determinant common to glutamate decarboxylase and coxsackie virus in insulin-dependent diabetes.

Insulin-dependent diabetes (IDD) results from the autoimmune destruction of the insulin-producing pancreatic beta cells. Autoreactive T-lymphocytes are thought to play a pivotal role in the pathogenesis of IDD; however, the target antigens of these cells, as well as the inductive events in the disease, are unclear. PBMC in persons with or at increased risk for IDD show elevated reactivity to the beta cell enzyme glutamate decarboxylase (GAD). To identify the T-lymphocyte-reactive determinants of GAD, an overlapping set of synthetic peptides was used to stimulate the PBMC from these individuals, PBMC responsiveness to GAD peptides was not restricted to those with IDD, and a number of peptides elicited responses in PBMC. However, the major determinant of GAD recognized by persons at increased risk for IDD was amino acids 247-279, a region which has significant sequence similarity to the P2-C protein of Coxsackie B virus (47% of 15 increased risk [islet cell autoantibody-positive relatives]; 25% of 16 newly diagnosed IDD patients; and 0% of 13 healthy control subjects). Responses to tetanus and insulin antigens were not different between the study groups. In addition, PBMC from individuals responding to GAD peptides within 247-279 also responded to a Coxsackie viral peptide (i.e., P2-C amino acids 32-47), an observation supporting potential molecular mimicry in this immune response. Although the role of environmental agents in the pathogenesis of the disease remains unclear, these cellular immunological findings support the epidemiological evidence suggesting an inductive role for enteroviruses like Coxsackie B in the autoimmunity underlying IDD.

Islet cell cytoplasmic autoantibody reactivity to glutamate decarboxylase in insulin-dependent diabetes.

Individuals with or at risk for insulin-dependent diabetes (IDD) frequently have autoantibodies against an islet cell cytoplasmic (ICA) antigen thought to be a sialoglycolipid. However, we now report that preabsorption of ICA-positive sera with recombinant glutamate decarboxylase (human GAD 65 and/or GAD 67) reduced or blocked the ICA reactivity of 5/18 (27%) new-onset IDD patients and 7/18 (39%) prediabetics. Interestingly, nondiabetic subjects with ICA of > or = 5 yr in duration had GAD-reactive ICA significantly more often (16/24, 67%, P < 0.04) than the diabetic groups. ICA reactivity to GAD was not related to serum ICA titer nor the age of the individual, and in all cases tested was blocked by GAD 65 or GAD 67 with equivalent efficiency. The ICA observed in 21/25 (84%) IDD patients with ICA long after clinical onset of disease (9-42 yr) was reactive to GAD. A natural history analysis of three individuals showed conversions from ICA which was reactive to GAD to a non-GAD-reactive ICA nearer to their clinical onsets of IDD. This study further defines the autoantigens reactive to ICA, and suggests that, whereas ICA that are not reactive to GAD may identify an advanced and more prognostic lesion, GAD-reactive ICA may typify the early or inductive lesion that may or may not progress to clinically significant beta cell injury.

Autoimmunity to two forms of glutamate decarboxylase in insulin-dependent diabetes mellitus.

Insulin-dependent diabetes mellitus (IDDM) is thought to result from the autoimmune destruction of the insulin-producing beta cells of the pancreas. Years before IDDM symptoms appear, we can detect autoantibodies to one or both forms of glutamate decarboxylase (GAD65 and GAD67), synthesized from their respective cDNAs in a bacterial expression system. Individual IDDM sera show distinctive profiles of epitope recognition, suggesting different humoral immune responses. Although the level of GAD autoantibodies generally decline after IDDM onset, patients with IDDM-associated neuropathies have high levels of antibodies to GAD, years after the appearance of clinical IDDM. We note a striking sequence similarity between the two GADs and Coxsackievirus, a virus that has been associated with IDDM both in humans and in experimental animals. This similarity suggests that molecular mimicry may play a role in the pathogenesis of IDDM.

How, when, and why to predict IDDM.

Insulin-dependent diabetes remains a serious disease replete with life-threatening complications. The onset of the disease is becoming increasingly predictable through the detection of its associated autoantibodies. The natural history of pathogenic events is attenuated in those with adult onset over that seen in infants and children. Immunosuppression therapies in newly diagnosed patients have been shown to induce remissions, whereas various intervention strategies in rodent models (BB rats and nonobese diabetic mice) can delay and even prevent the onset of diabetes. The stage is set for serious consideration of intervention trials to delay the clinical disease in humans.

Autoantibodies in nonobese diabetic mice immunoprecipitate 64,000-Mr islet antigen.

In insulin-dependent diabetes mellitus (IDDM) in humans and BB rats, islet cell autoimmunities associated with autoantibodies to a beta-cell protein of 64,000 Mr (64K) have been described. We report that sera from newly diagnosed nonobese diabetic (NOD) mice similarly contain an autoantibody that immunoprecipitates 64K autoantigen from detergent lysates of [35S]methionine-labeled murine islet cells. The autoantibody was detectable by weaning; it disappeared within weeks after diabetes onset and was absent in older nondiabetic NOD mice as well as all of three non-diabetes-prone control strains tested. The 64K beta-cell autoantigen may be a critical target in the immunopathogenesis of IDDM.

Antibodies to nucleic acids in juvenile-onset diabetes.

Antibodies to single stranded (SS-) and double stranded (DS-) DNA and RNA were determined by a passive microhemagglutination assay in sera from 80 children with juvenile-onset diabetes mellitus (JDM) and 129 children with asthma. The latter group was chosen for comparison with the JDM group because of their increased susceptibility to viral infection and the nonautoimmune nature of the disease. We found that JDM patients had increased titers of antibodies to SS-DNA (61.3%), synthetic polyadenylicpolyuridylic acid (Poly A-U) (78.8%), synthetic polyinosinicpolycytidylic acid (Poly I-C) (62.5%), and DS-RNA of statolon virus (51.3%) and reovirus (27.3%), respectively, in contrast to asthmatics (15.5, 34.9, 3.9, 20.2, and 2.3%, respectively) or to healthy controls. The difference of the incidence of antibodies among the groups is statistically significant (P less than 0.001). Presence of SS-DNA antibody found in two thirds of cases of JDM further support the increased prevalence of autoimmune phenomenon in that disease. Furthermore, the increased prevalence of DS-RNA antibodies in patients with JDM, found especially in cases of recent onset, is suggestive of an active immune response against the underlying viral replications that may have led to beta cell injury in islets of pancreas.

Insulitis and diabetes in NOD mice reduced by prophylactic insulin therapy.

Intensive insulin therapy in patients with recently diagnosed insulin-dependent diabetes mellitus (IDDM) has been reported to result in a prolonged increase in endogenous insulin-secreting capacity. Because the clinical onset of IDDM occurs only after most insulin-secreting beta-cells have been destroyed, we tested whether prophylactic insulin therapy might prevent IDDM in nonobese diabetic (NOD) mice. One hundred fourteen NOD mice were randomized at weaning into a protamine zinc pork insulin-treated (I) group or a placebo-treated (P) group given insulin diluent. All insulin treatments were adjusted to the maximum tolerable dosages and continued until 180 days of age. The cumulative IDDM frequency within the female I group was significantly less (3 of 34, 8%) than in female P controls (17 of 26, 65%; P less than 0.0001). This beneficial effect was limited to females, however, because the frequency of IDDM in male I mice (3 of 32, 9%) was not significantly different from the frequency in male P controls (1 of 22, 5%; P less than 0.5). Pancreatic histological examinations of nondiabetic animals revealed that insulin treatment resulted in significant reductions in islet cell inflammation and damage and improvements in insulin content. In summary, NOD mice given insulin therapy from weaning until 180 days of age had significantly lower frequencies of diabetes and pancreatic insulitis than sex-matched control littermates treated with insulin diluent. These results suggest that prophylactic insulin therapy to prevent IDDM in humans should be considered for clinical trials.

Predictive value of gastric parietal cell autoantibodies as a marker for gastric and hematologic abnormalities associated with insulin-dependent diabetes.

The frequency and significance of gastric parietal cell autoimmunity was assessed in 771 patients with insulin-dependent diabetes (IDD) of onset before 30 yr of age. Gastric parietal autoantibodies (PCA) were found 4 times more frequently in the patients with IDD (9%) than among 600 matched nondiabetic controls (2%). Caucasian female patients with IDD had PCA twice as frequently as male patients. Thyroid microsomal autoantibodies were more frequent in patients with IDD and PCA, than in those with IDD alone (Caucasian 46% versus 18%, black 25% versus 2.5%). A history of pernicious anemia and/or PCA was found in 25 or 40 families of IDD probands with PCA. Achlorhydria was demonstrated in 6 of 11 patients (54%) with PCA but in none of seven IDD patients without PCA. The six patients with achlorhydria had significantly lower uptakes of oral radiolabeled cobalamin, lower serum cobalamin levels, lower intrinsic factor-R protein ratios in their gastric aspirates, and lower plasma ferritin levels than patients with IDD but without PCA. None of the study group had IF antibodies in their serum or gastric juice. Overt pernicious anemia and neuropathy were found in one patient with PCA. Young patients with IDD at risk for atrophic gastritis and cobalamin deficiency can initially be identified by screening for PCA. Many of these young patients with PCA already have achlorhydria and evidence of decreased absorption of cobalamin. These patients can then be followed with cobalamin levels and/or with complete blood counts to identify those requiring therapy.

Ten-year prognosis of impaired glucose tolerance in siblings of patients with insulin-dependent diabetes.

We traced 105 of 140 siblings of children with insulin-dependent diabetes (IDD) who had had oral glucose tolerance tests (OGTT) 10-12 yr earlier. Siblings with abnormal tests by screening criteria (8.3 mmol/L at 1 h, 7.2 at 2 h, N = 44) included all 6 who subsequently developed IDD after 3 mo to 7 yr (5.7% of entire group, 13.6% of abnormal screenees). The National Diabetes Data Group criterion for children (7.8 mmol/L at 2 h) identified 19 siblings, including 5 of the 6 who later developed IDD (26% of abnormals). Subsequent full 4-h OGTT, including analysis of insulin responses, did not improve predictability for subsequent IDD. Thus, siblings of IDD were identified at high risk (14-26%) or at low risk (0-1%) for subsequent IDD by a simple 2-h OGTT. The prolonged latency in the development of IDD indicates that, among siblings of IDD, this disorder may be already chronic for years by the time of clinical onset.

Pancreatic alpha cell autoantibodies and glucagon response to arginine.

The frequency and significance of cytoplasmic pancreatic alpha cell autoantibodies (ACA) were investigated in 2102 healthy controls, 879 patients with insulin-dependent diabetes mellitus (IDDM) who were negative for islet cell autoantibodies (ICA), and 1567 relatives of IDDM patients. ACA were found in approximately 1 in 200 people of all ages and were not significantly associated with IDDM, the IDDM-associated HLA phenotypes DR3 and DR4, or thyrogastric or adrenal autoantibodies. Of 11 ACA-positive patients studied by arginine stimulation tests, none had frank glucagon deficiency. Thus, ACA do not appear to be associated with defective alpha cell function or with IDDM.

The diabetogenic effects of streptozotocin in mice are prolonged and inversely related to age.

Single "subdiabetogenic" doses of streptozotocin (SZ), when given to young male CD-1 mice, produced a delayed onset of hyperglycemia dependent on the dose of SZ and on the age of the mice. The effect was markedly reduced or absent in older mice given the same dose of SZ per kg of body weight. Histologic examination of the pancreas of these animals revealed that SZ induced greater damage to the islets of the young mice compared with older mice. In addition to the characteristic findings of a decrease in insulin-containing cells and an increase in glucagon- and pancreatic polypeptide-containing cells there was evidence of new islet formation. Delayed-onset hyperglycemia was also induced in young inbred DBA/2J, C57BL/KsJ, and SWR/J mice with single SZ doses as well as with alloxan in young CD-1 mice, indicating that the effect was not specific for CD-1 mice not for SZ as the agent inducing beta-cell injury. The induction of beta-cell autoimmunity did not appear to be important in the delayed diabetogenic effect of SZ, since insulitis was rare and followed the onset of hyperglycemia when seen, and islet cell autoantibodies were not found. Rather, SZ induced more beta-cell destruction in young animals than in older mice, and the continued somatic growth of the former suggests that the delayed hyperglycemia was due to an out-growing of a reduced insulin supply. That mild to severe diabetes could be induced by the same dose of SZ/kg, depending only on the age of the mice when SZ was given, may have implications for understanding the apparent heterogeneity of human diabetes mellitus.

Lymphocyte subsets and activation in prediabetes.

Peripheral blood lymphocytes were obtained from 65 individuals: 34 nondiabetic patients with islet cell autoantibodies (ICA) (prediabetic phase), 9 patients with newly diagnosed insulin-dependent diabetes mellitus (IDDM), 6 ICA-negative siblings or offsprings of IDDM patients, and 16 ICA-negative controls. The presence of lymphocyte abnormalities and/or activation was examined with dual fluorescence flow cytometry. The percentages of B cells, total T-lymphocyte, and helper T-lymphocyte (Th) and cytotoxic/suppressor T-lymphocyte (Tc/s) subsets and their ratio were not significantly different among the patient groups. No increased expression of interleukin 2 receptor on T-lymphocyte was found in newly diagnosed IDDM or prediabetic individuals. Sixteen of 49 patients had significantly increased number of T-lymphocyte expressing HLA-DR. A significant increase in the number of both Th and Tc/s subsets expressing HLA-DR was found in only 3 of 16 patients. This increase was unrelated to the patients's relative ICA titer or HLA-DR phenotype. On the other hand, the relative density of the DR antigen (RAD-DR) was significantly increased on both Th (886 +/- 120) and Tc/s (1250 +/- 273) in 13 of 38 patients compared with control patients (Th 484 +/- 129 and Tc/s 460 +/- 166). The RAD-DR on Tc/s correlated with the relative ICA titer and was greatest on DR3/4-phenotyped T-lymphocytes. In addition, significantly increased RAD-DR was found in noncontrol patients with impaired insulin release responses at 1 and 3 min to intravenous glucose.(ABSTRACT TRUNCATED AT 250 WORDS)

Restriction-fragment-length polymorphisms of 5'-flanking region of insulin I gene in BB and other rat strains. Absence of association with IDDM.

Because the 5'-flanking hypervariable region of the human insulin gene may be associated with insulin-dependent diabetes mellitus (IDDM), we examined the spontaneously diabetic BB rat and other rat strains for polymorphisms of the two rat insulin genes, the localization of such polymorphisms, and their possible association with IDDM. By use of restriction-fragment-length polymorphism analysis, we found that the transcribed portion of the insulin I gene, its 3'-flanking region, and the insulin II gene were not polymorphic. However, four alleles of the insulin I gene were identified, two of which (IA and IB) were found in BB rats. Alleles IA and IB varied in their 5'-flanking regions, yet neither was associated with IDDM in the BB rat.

Competitive insulin autoantibody assay. Prospective evaluation of subjects at high risk for development of type I diabetes mellitus.

A quantitative fluid-phase radioassay for autoantibodies reacting with insulin (competitive insulin autoantibody assay, CIAA) was developed. The assay's features include 1) use of a physiologic amount of 125I-labeled insulin, 2) parallel incubations with supraphysiologic cold insulin (competitive), and 3) an incubation time of 7 days and a single-step multiple-wash polyethylene glycol separation. Mean +/- SE CIAA levels in 50 controls were 8 +/- 1.4 nU/ml (range -16-33.3). In 36 cytoplasmic islet cell antibody (ICA)-positive nondiabetic first-degree relatives of type I (insulin-dependent) patients less than 30 yr of age, CIAA levels exceeded the normal range in 20 (55.6%) of 36 (mean 86.8 +/- 17.1 nU/ml). In 26 ICA-positive relatives greater than 30 yr of age, only 5 (19.2%) of 26 exceeded the normal range (mean 26.1 +/- 9.4 nU/ml); P less than .001 compared with younger ICA-positive relatives). Six ICA-negative HLA-identical siblings of type I diabetic patients had normal CIAA levels (mean 3.6 +/- 5.8 nU/ml), and only 2 of 13 ICA-negative identical twins discordant for diabetes (mean 15.4 +/- 6.6 nU/ml) exceeded the normal range. Nine (50%) of 18 ICA-positive schoolchildren exceeded the normal range (mean 105.3 +/- 36.7 nU/ml). Genetically susceptible subjects negative for CIAA (with only 3 exceptions) remained negative for CIAA on multiple determinations (3 conversions observed), and CIAA levels of positive subjects were relatively stable. Linear regression of the first CIAA level versus last (interval between sampling 1 mo to 10 yr) in genetically susceptible individuals showed a highly significant correlation (r = .95, P less than .001).(ABSTRACT TRUNCATED AT 250 WORDS)

Improved specificity of ICA assays in the Fourth International Immunology of Diabetes Serum Exchange Workshop.

The goal of the Fourth International Workshop for Standardization of ICA Measurements was to determine the specificity of ICA assays and their ability to distinguish between control sera (n = 57) and sera from IDDM-related individuals--representing relatives of IDDM patients (n = 21), healthy individuals who later developed IDDM (n = 8), or newly diagnosed IDDM patients (n = 23). Results from 28 laboratories were analyzed. The mean specificity (percentage of control sera reported as negative) among 27 laboratories was 91%, including 6 laboratories with 100% specificity. Nevertheless, 78% of laboratories found at least one control sample > 0 JDF U. Among samples from first-degree relatives, the mean concordance was 86%, including three sera found negative (0 JDF U) by all laboratories. Among individuals who later developed diabetes, the mean concordance was 93%, with two sera found positive by 100% of laboratories. In sera from newly diagnosed IDDM patients, the mean concordance was 82%. Three sera were found positive and one serum negative by all laboratories. The JDF U of the sera considered to be positive were significantly greater than each laboratory's average for the controls. In conclusion, the results from laboratories participating in the Fourth International ICA Workshop demonstrated excellent specificity, good concordance, and an ability to separate control sera from defined, IDDM-related subjects.