Investigations of humans and nonobese diabetic mice suggest that proinsulin and/or a fragment of the region spanning C-peptide and the B-chain of insulin (i.e., proinsulin peptide) may serve as key autoantigens in IDDM. Therefore, we analyzed cellular immune reactivities against these molecules in people with or at varying risks for the disease to clarify their role in the pathogenesis of IDDM. In vitro peripheral blood mononuclear cell (PBMC) responses against these antigens, a control antigen (tetanus toxoid), and phytohemaglutinin were determined in 60 individuals with newly diagnosed IDDM (< or = 1 day from diagnosis) in 34 islet cell cytoplasmic autoantibody- and/or insulin autoantibody-negative first-degree relatives of the IDDM subjects, and in 28 autoantibody-negative control subjects. Unlike previous reports suggesting diabetes-associated elevations in cellular immunity to other beta-cell antigens (e.g., GAD, IA-2, etc.), we observed equivalent levels of phytohemaglutinin stimulation and cellular proliferation in all groups against these antigens (all P values were not significant). The mean stimulation index +/- SD and frequency of reactivity to proinsulin for healthy control subjects and IDDM patients, respectively, were as follows: 1 microg/ml (1.5 +/- 1.0, 1 out of 17 [6%]; 1.9 +/- 1.4, 4 out of 33 [12%]); 10 microg/ml (1.7 +/- 1.3, 1 out of 17 [6%]; 1.2 +/- 0.6, 0 out of 28 [0%]); and 50 microg/ml (1.2 +/- 0.6, 1 out of 16 [6%]; 1.1 +/- 0.6, 1 out of 27 [4%]). The response in healthy control subjects, autoantibody-negative relatives, and IDDM patients, respectively, against the proinsulin peptide fragment were as follows: 1 microg/ml (0.9 +/- 0.4, 1 out of 12 [8%]; 1.3 +/- 1.1, 4 out of 34 [11%]; 1.1 +/- 0.3, 2 out of 28 [7%]); 10 microg/ml (0.9 +/- 0.6, 1 out of 12 [8%]; 1.2 +/- 0.6, 3 out of 34 [9%] 1.4 +/- 1.7, 2 out of 28 [7%]); and 50 microg/ml (1.0 +/- 0.7, 1 out of 12 [8%]; 1.2 +/- 0.5, 2 out of 34 [6%]; 1.3 +/- 0.5, 2 out of 28 [7%]). Taken together with previous studies reporting relatively infrequent occurrences of autoantibodies to proinsulin, the role of immunity to this molecule in the pathogenesis of IDDM in humans remains unclear.

Type I diabetes mellitus appears as the end result of an autoimmune destruction of the insulin secreting pancreatic beta cells [1]. The immunological mechanisms underlying the development of Type I diabetes mellitus as well as the role of environmental agents (e. g. diet, viruses, stress) in the pathogenesis of the disease have been the subject of considerable research. The rationale for investigating environmental agents involves an attempt to explain numerous epidemiological observations including the disease discordance rate amongst twins with Type I diabetes mellitus, a pronounced north-south gradient in the incidence of Type I diabetes mellitus, reports of an increased (e. g. up to fourfold) incidence of Type I diabetes mellitus over the past three decades, and the estimate that more than 80 % of patients with newly-diagnosed Type I diabetes mellitus do not have a firstdegree relative with the disease [2]. Diabetologia (1998) Summary Elevated cellular immune responses against the cows' milk protein b casein have been reported in individuals with Type I diabetes mellitus, a finding supportive of the concept that cows' milk consumption may be causative for the disease. We analysed cellular immune reactivities against b casein in newly-diagnosed Type I diabetic patients, their immediate autoantibody negative relatives, and unrelated healthy individuals in order to further elucidate the role of anti-b casein immunity in the pathogenesis of Type I diabetes mellitus. Peripheral blood mononuclear cells were stimulated in vitro with various concentrations of three different b casein preparations, control antigens (tetanus toxoid, mumps extract) and a mitogen (phytohemagglutinin). The frequency and/or mean simulation index of cellular proliferation against two of the b casein preparations at high antigen concentrations (i. e. 10 or 50 mg/ml) were significantly higher in newly-diagnosed Type I diabetic subjects compared with autoantibody negative healthy control subjects. However, reactivities against b casein in the Type I diabetic probands and their autoantibody negative relatives, individuals with a very low-rate of disease development, were almost identical. Cellular immune reactivities to other antigens were similar between the subject groups. In addition to indicating the need for appropriately matched subject populations (e. g. human leukocyte antigen (HLA) matched relatives) when analysing cellular immune responses, these findings support our previous contention that individuals genetically prone to autoimmunity may be deficient in forming tolerance to dietary antigens. However, the significance of anti-b casein immunity as a specific causative factor in the pathogenesis of Type I diabetes mellitus remains unclear.

The period of efficacious immune reactivity afforded by tetanus immunization and the need for continuing some forms of tetanus vaccination programs have been the subjects of recent debates. Our studies demonstrate that the level of antitetanus immunity based on immunological memory (i.e., cellular immune responsiveness) varies dramatically as a function of age, with older individuals constituting a population which is increasingly susceptible to tetanus infection.

Large-scale programs designed to assess risk for type 1 diabetes through serologic assessment of autoantibodies to recombinant beta-cell autoantigens are hampered by several limitations, including the methods for sample collection and assay performance, as well as the volume required for autoantibody determinations. The present study was designed to develop a low sample-volume, primary screening method for autoantibody detection of high specificity and sensitivity, and to determine the feasibility of dried blood spots collected on filter paper in serving as vehicles for such determinations. Autoantibodies to glutamic acid decarboxylase (GAD) and ICA512bdc (IA-2), both individually and in combination, were determined in persons with type 1 diabetes, healthy controls, or individuals with other autoimmune disorders. Autoantibody results for serum, plasma, and dried blood spots were compared. GAD, IA-2, and combined GAD/IA-2 autoantibodies were concordant in their measurement from minimal volumes of serum, plasma, and whole blood extracted from dried filter paper. The autoantibody levels from the dried blood spots were, however, lower than corresponding serum samples, and, as currently designed, failed to detect low-titer autoantibodies. Despite this limitation, screening for diabetes risk can be performed using small volumes of whole blood, serum, or plasma collected onto filter paper. These methodological improvements should simplify matters, reduce costs, and increase the efficacy of screening programs for type 1 diabetes. Further development of better substrates/methods for blood-specimen collection seems necessary to exploit the full potential of this and other autoantibody measurement strategies for screening large populations.