The height, weight and menarche of children and young people treated in the Children's Clinic as in-patients in 1987 was determined in dependence on the age at onset of diabetes and the duration of the disease, and the frequency of the disturbances of somatic development were shown. A growth retardation was registered in 1.8% only while the frequency of obesity amounted to 10%; 13.9% in female and 6.3% in male type I diabetics. However, the overall percentage of obesity in diabetics does not differ significantly from that in healthy subjects of corresponding age. With a higher age of onset, the frequency of adiposity increased significantly in both sexes; the highest frequency was shown by the girls who contracted the disease between the ages of 10 and 14. While the frequency of adiposity decreased significantly in male patients with increasing duration of diabetes, a non-significant increase was observed in female probands. There was no connection between adiposity and insulin dosage. The mean age for menarche in diabetic girls was determined at 13.4 years. The greatest, non-significant delay was experienced by those where onset of diabetes was between the ages of 5 and 9. The reason for this retardation is presumably the quality of the metabolism.

Serum activities of complement-dependent antibody mediated cytotoxicity (C'AMC) were determined in 36 consecutive patients with newly diagnosed insulin-dependent diabetes mellitus (IDDM). The sequential exposure of 51Cr labeled neonatal rat islet cells to patient serum and rabbit complement revealed the presence of C'AMC in 28 IDDM subjects. The C'AMC titres ranged between 1:4 and 1:512 and were not related to the C'AMC activity of a given sample as measured at a standard dilution (1:4). In comparison to the clinical characteristics of 21 IDDM patients with negative C'AMC, higher C'AMC titres (greater than or equal to 1:32) were associated with a lower mean age at diagnosis of IDDM (12.2 +/- 2.1 vs. 19.0 +/- 2.3 years; p less than 0.05), with a higher frequency of infections up to 6 months prior to diagnosis (6 out of 11 vs. 3 out of 21 patients; p less than 0.05) and, although statistically not significant, a preponderance of female sex together with a decreased frequency of HLA-DR4. In contrast, fasting C-peptides levels, HLA-DR3 antigen frequency and Coxsackie B1-6 virus antibody titres were not related to the C'AMC titres. It is concluded that (1) C'AMC titration is superior to the detection of initial C'AMC levels for evaluating the strength of the complement-dependent humoral immune response towards islet cell surface (auto)antigen(s), and (2) infectious agents may be involved in eliciting a C'AMC response.

In a two-year follow-up study neonatal rat islets have been shown to be affected in vitro by lymphocytes and complement-inactivated serum obtained from newly diagnosed Type 1 (insulin-dependent) diabetic patients and probands who are at high risk for developing the disease. The effect was measured by 51Cr-release of the islets treated with the proband's serum after a 6 h-incubation with lymphocytes of the same donor. Nineteen newly diagnosed diabetic patients, 23 persons at risk and 11 control probands were studied. There was no appreciable cytotoxic activity in the control probands (with one exception) and in 7 out of the 19 newly diagnosed diabetics. Five of the diabetes-susceptible probands developed diabetes mellitus during the investigation period. Anti-islet cytotoxicity of lymphocytes was found in these individuals at least 8 months before diagnosis of Type 1 diabetes. The cytotoxic effect disappeared at various time intervals after disease manifestation. Islet cytotoxicity was intermittently found with lymphocytes from further 13 probands at risk, sometimes for more than one year. Our data indicate that mononuclear cells from probands who are at high risk for developing Type 1 diabetes can exert cytotoxicity on xenogenic neonatal islets in the presence of their own serum.

In 868 insulin-treated diabetic children and adolescents with onset of IDDM under age 20 we investigated the frequency of IDDM and NIDDM in all first-degree relatives. On the basis of the National Diabetes Register of the GDR the age-corrected lifetime risk for the development of IDDM and NIDDM was calculated for the general population and for parents and siblings of diabetic children. The age-corrected risk for IDDM, but not for NIDDM, is statistically significantly higher in fathers (10.26 +/- 1.75%) than in mothers (5.28 +/- 1.49%) and is about equal in brothers (30.71 +/- 6.07%) and sisters (35.54 +/- 6.28%) of children with IDDM. Among general population the age-corrected life-time risk for IDDM is equal for males (4.35 +/- 0.02%) and females (4.91 +/- 0.02%), but is significantly higher for NIDDM in females (27.49 +/- 0.04% contrary to 24.07 +/- 0.05%). In comparison with the data of Tillil and Köbberling (1987) our lifetime risk estimates show a shifting of risk for IDDM and NIDDM into older age groups.

Autoantibodies against insulin, C-peptide, and glucagon were determined by radio-binding assay in 63 new-onset Type 1 (insulin-dependent) diabetic patients as well as in 70 controls. Plasma peptide binding was determined by means of 125I-labeled peptides and charcoal-dextran separation technique. Binding values exceeding the mean plus three standard deviations of the controls were considered as antibody-positive. Sixteen patients (25%) were positive for IAA, as 6 (10%) were positive for CAA and 2 (3%) for GAA. Of all control subjects, none were positive for either IAA or CAA, whereas 2 (2%) had GAA. The mean 125I-glucagon binding in the patients' group was, however, slightly enhanced and could be suppressed to normal values by excess unlabeled glucagon. The presence of IAA and/or CAA was significantly associated with more severe symptoms at diabetes manifestation. These results indicate that in new-onset Type 1 diabetics autoimmunity arises against all the insular peptides tested but is predominantly directed against those antigens secreted from the beta cells. Nevertheless, extremely low-binding GAA seem to be common in these patients. The determination of IAA/CAA might be useful in detecting a possible heterogeneity of Type 1 diabetes with regard to its clinical mode of manifestation.

In 30 first-degree relatives (siblings or children) of type-I diabetics and 17 relatives (children) of type-II diabetics as well as in 19 healthy subjects a two-hour glucose infusion test (GIT, 12 mg/kg b.w./min) primed by a starting bolus of 0.33 g/kg b.w. was performed to evaluate carbohydrate tolerance (CHT) and insulin secretion pattern. After 4 to 5 years the test was repeated and the results were compared with those of the initial GIT. The glucose-stimulated insulin response of the early secretion phase (0--5 min) decreased during the follow-up study in relatives of type-II diabetics with normal CHT (in tendency) and with glucose intolerance (p less than 0.05) but not in relatives of type-I diabetics. A rightward shift of the glucose-insulin response curve was seen in the former group. Relatives of type-II diabetics with impaired CHT showed a striking abnormality in B-cell responsiveness. In relatives of type-I diabetics a disturbed glucose-insulin response curve could not be observed. The insulin response during the late phase of insulin secretion did not differ significantly among the groups. The conclusion drawn from our findings is that responsiveness of pancreatic B-cells seems to be directly affected genetically in first-degree relatives of type-II but not of type-I diabetics. Thus, diabetes mellitus cannot be regarded as one disorder with a similar genetic background.

UNLABELLED:Present cross-sectional clinical study was aimed at the evaluation the prevalence of cardiovascular risk factors in Type 2-diabetics suffering from different clinical manifestations of diabetic foot lesions due to peripheral vascular disease and/or diabetic neuropathy. 1025 non-insulin-dependent (Type 2) diabetics (NIDDM) of both sexes were investigated. Patients were classified in Type II diabetes without peripheral vascular disease and foot lesions (group 0, controls), with macroangiopathic related foot lesions (group 2), with neuropathic foot lesions (group 3), and with mixed neuropathic-ischemic foot lesions (group 4). Apart from urinary albumin excretion rate (UAE), the following micro- and macroangiopathic risk factors and diseases were taken into account: Hypertension, degree of metabolic control (HbA1c), lipid concentrations, duration of diabetes, retinopathy, clinical nephropathy.RESULTS:In the total population the UAE was significantly (p < 0.01) correlated with duration of diabetes, serum creatinine, hypertension, age, lipid concentrations, HbA1c and insulin requirement. In comparison to Type II diabetic patients without peripheral vascular disease (group 0) and with neuropathic foot lesions (group 3), subjects with ischemic (group 2) and mixed neuropathic-ischemic foot lesions demonstrated an increased prevalence of pathological UAE, which was associated with a higher frequency of clinical nephropathy, retinopathy, an older age and longer duration of diabetes. It is concluded that microalbuminuria in Type 2 diabetes reflects both the existence of diabetic nephropathy and peripheral vascular disease which is often associated with the insulin resistance syndrome.

It has been suggested that the mumps virus may be involved in the etiopathogenesis of Type-I diabetes mellitus. Most studies have analyzed this relationship retrospectively. We, however, carried out a prospective study over a 4 year period after a mumps infection in two age groups (16 years and under [group A no = 32] and over 16 years [group B no = 18]). These subjects with "diabetic risk factors" (impaired glucose tolerance, low insulin response, ICSA and/or HLA-DR3/DR4) were selected from 1581 registered cases, in whom an antecedent mumps infection had occurred in 1980 and 1981. Glucose tolerance and insulin secretion did not change significantly during 4 years after a mumps infection. Overt diabetes was not observed in any of the cases. One year after a mumps infection 35% of children and 63% of adolescents/adults exhibited ICSA (control subjects = 5%; a serum was considered ICSA-positive if more than 25% of the intact rat islet cells showed distinct cell surface immunofluorescence). After 4 years the percentage of subjects with ICSA decreased significantly to 13% and 14%, resp. Only 21% of ICSA-positive sera were found to be cytotoxic on rat islet cells (51Cr-release assay). No relationship could be evaluated between complications resulting from a mumps infection and the appearance of ICSA. There was no correlation between ICSA, glucose tolerance, and insulin secretion. In fact, our prospective study did not reveal any relationship between a mumps infection and Type-I diabetes. ICSA would seem to be of no predictive value.

Polymyositis (PM) and dermatomyositis (DM) are the prototypical inflammatory diseases of skeletal muscle. In PM, CD8+ T cells invade and destroy muscle fibers, whereas humoral effector mechanisms prevail in DM. We studied the expression of the cytotoxic mediator perforin in inflammatory cells in PM and DM muscle by semiquantitative PCR, immunohistochemistry and confocal laser microscopy. Similar levels of perforin mRNA were expressed in PM and DM, and abundant perforin-expressing CD3+CD8+ and CD3+ CD4+ T cells were observed in both diseases. However, there was a striking difference in the intracellular localization of perforin. In DM, perforin was distributed randomly in the cytoplasm of the inflammatory T cells. In contrast, 43% of the CD8+ T cells that contacted a muscle fiber in PM showed perforin located vectorially towards the target muscle fiber. The results suggest (a) that the random distribution of perforin in the cytoplasm of muscle-infiltrating T cells observed in DM reflects nonspecific activation, and (b) that the vectorial orientation observed only in PM reflects the specific recognition via the T cell receptor of an antigen on the muscle fiber surface, pointing to a perforin- and secretion-dependent mechanism of muscle fiber injury.

Cytoplasmic islet cell antibodies, glutamic acid decarboxylase autoantibodies, spontaneous insulin autoantibodies, and insulin-induced antibodies were analyzed in a 1-year follow-up study of 12 newly diagnosed patients with insulin-dependent diabetes mellitus aged 14 +/- 2 years (range 7-20 years) who had been initially treated with either multiple injections of insulin alone (control group) or, in addition, anti-CD4 monoclonal antibody/prednisolone (treatment group). Despite individual variations in islet cell antibody titers, there were no significant differences in the prevalence or changes in the mean titers between the two groups. Glutamic acid decarboxylase autoantibodies remained almost unchanged, but correlated with levels of islet cell antibodies. While at initiation of treatment only 50% of the patients from both groups had spontaneous insulin autoantibodies, all patients developed insulin-induced antibodies upon conventional insulin therapy during the course of follow-up. This was not related to islet cell antibody or glutamic acid decarboxylase antibody levels. The insulin requirement was markedly reduced through the period of follow-up, but did not significantly differ between the two groups. A correlation between islet cell antibody levels and insulin requirement was observed in the control group but not in the treatment group. Plasma levels of the antibodies were not associated with changes in stimulated C-peptide or hemoglobin A1 concentrations. Activated T-lymphocytes persisted in both groups of patients, but their mean levels were not significantly different. The reason for the absence of statistically significant differences between treatment and control groups could be due to the small number of patients in the study. In conclusion, short-term immune intervention with anti-CD4 monoclonal antibody in addition to insulin therapy did not suppress autoimmune reactions towards the beta cells.

Increased erythrocyte sodium-lithium countertransport activity has been implicated in the pathogenesis of diabetic nephropathy. However, its relationship to other cation membrane transport systems in incipient nephropathy is not yet clear. The present study was thus performed to: (1) explore associations between sodium-lithium countertransport and changes in the activity of other cation transport pathways and (2) to compare the sodium transport activities with clinical characteristics of insulin-dependent diabetic patients with and without evidence of incipient diabetic nephropathy. We measured erythrocyte sodium-lithium countertransport, passive sodium/ potassium flux (at 1°C), adenine nucleotide content in intact erythrocytes and sodium/potassium-, magnesium-and calciumdependent ATPase activity in erythrocyte membrane preparations from 34 insulin-dependent diabetic patients without microalbuminuria, 8 diabetic patients with microalbuminuria, and 8 age-matched healthy control subjects. Sodium-lithium countertransport was elevated in diabetic patients with normo- and microalbuminuria compared with control subjects [268±99 and 299(277–465), respectively, vs. 166±65 μmol/(l cells×h)] and was positively correlated (r=0.36,P<0.05) with the albumin excretion rate. However, the activity of erythrocyte membrane ATPases was significantly decreased compared with control subjects. The ATP and ADP content was found to be significantly higher (P<0.001) in erythrocytes from diabetic patients compared with control subjects (1,196±276 vs. 833±253 μmol/l cells and 353±97 vs. 255±64 μmol/l cells, respectively). The extent of erythrocyte potassium leakage correlated with hemoglobin A1c (r=0.39,P<0.05). These results demonstrate that changes in the activity of membrane cation transport, occurring in early nephropathy, are not confined to sodium-lithium countertransport, but involve various pathways, thus reflecting underlying membrane alterations which are at least partially influenced by the diabetic milieu.

Two- and three-color cytofluorimetric techniques were used to study the expression patterns of the activation antigen HLA-DR on peripheral blood immunoregulatory T-cells from 25 patients with newly diagnosed insulin-dependent diabetes mellitus (IDDM) and 14 age- and sex-matched control subjects. The mean percentage of total activated (CD3+HLA-DR+) T-cells was significantly elevated in the IDDM group compared with the control group (P < 0.001). In control subjects, basal activation of CD4+ and CD8+ lymphocytes accounted for the low percentage levels of activated T-cells. In contrast, the majority of IDDM patients showed an unbalanced activation of CD4+ and CD8+ lymphocytes with predominant activation of the CD8+ lymphocyte subset. The composition of the activated T-cell fraction was dependent on the composition of the total (activated + nonactivated) T-cell population, as indicated by the positive correlation between the CD4+/CD8+ T-cell ratios in these two cell populations (r = 0.714; P < 0.001). Excessive activation of CD8+ T-cells was attributable to similar increases in the proportions of CD8+CD45RA+HLA-DR+ (naive) and CD8+CD45RA−HLA-DR+ (memory) cells. Analysis of the CD11b-defined subsets revealed predominant activation of CD8+ CD11b− (cytotoxic) T-cells; CD8+CD16+ HLA-DR+ natural killer cells wereunchanged. The distribution of HLA-DR+ cells among subsets of CD4+ T-cells differed from the pattern in the CD8+ population in that selective activation of CD4+ CD45RA− (memory, helper-inducer) cells accounted for the small increase in activated CD4+ cells. The present findings suggest an important role for CD8+ T-lymphocytes in IDDM and raise the possibility that these lymphocytes might be the relevant target for early immunotherapy in patients with recent disease onset.

Several recent studies have shown that some inflammatory myopathies are autoimmune diseases. It is possible that certain alterations in the muscle-immune cell microenvironment and in the local production of cytokines could take part in the pathogenesis of inflammatory myopathies. In the present study we investigated the effects of tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) on the secretion of interleukin-6 (IL-6) by myoblasts. Purified human myoblasts from normal subjects and from patients with polymyositis were cultured in the presence of TNF-alpha and IFN-gamma at two concentrations (100 and 200 U/ml), alone or in combination, for 12, 24 and 48 h. The supernatants were collected and the IL-6 concentrations tested by ELISA (Genzyme). We found that myoblasts secrete IL-6 constitutively. The secretion of IL-6 was greatly increased by TNF-alpha; the increase was both time- and dose-dependent. IFN-gamma caused a moderate increase in IL-6 secretion, but this effect was not significant, despite a slight positive trend over time. There was no synergism in the effect of IFN-gamma and TNF-alpha.It is known that inflammatory myopathies ate characterized by mononuclear cell infiltration and muscle regeneration: myoblasts are present in infiltrated tissues. Thus, the local production of cytokines that characterizes the inflammatory reaction, could stimulate myoblasts to secrete IL-6, which might add to the pro-inflammatory effects of IL-6 produced by activated macrophages and T cells.

Two monoclonal antibodies specifically recognizing the 65 kDa isoform of the enzyme glutamic acid decarboxylase (GAD) were generated by fusion of spleen cells of a non-obese diabetic (NOD) mouse which had received a single intraperitoneal injection of 0.2 ml complete Freund's adjuvant followed three days later by one administration of a subdiabetogenic dose of streptozotocin (80 mg/kg body weight) three days before the fusion experiment was performed. Both monoclonals belong to the IgG1 isotype and were screened with an enzyme-linked immunosorbent assay using rat brain extract as a natural source of GAD and additionally with a capture assay by means of immunoglobulins of a patient with Stiff-man syndrome. The specific binding to the 65 kDa isoform of the enzyme was detected by a radioligand and an enzyme-linked immunosorbent assay using recombinant human glutamic acid decarboxylase specific for both the 67 and 65 kDa isoforms. Both monoclonal antibodies recognize the same antigenic epitope, which is located in the N-terminal region of the first 17 amino acids detected by fragments of human pancreatic 65 kDa GAD. Three out of 30 sera from Type 1 diabetic patients specifically displaced the binding of the monoclonals from 125I-labelled GAD65 measured by radio-immunoassay. A striking binding of both monoclonals M61/8F9 and M61/7E11 to the islets of cryosections of human, monkey, pig and rat pancreas but not to mouse pancreas was detectable. The antibodies failed to bind on the cell surface of viable rat islet cells. It is concluded that also in the diabetes-prone NOD mice GAD65 autoantibodies occur although GAD65 was not detectable in the mouse islets.

Serological findings have suggested that antibodies (Ab) to bovine serum albumin (BSA-Ab) are associated with type 1 diabetes mellitus. The aim of our study was to evaluate a competitive fluid-phase radioimmunoassay for detecting BSA-Ab using different incubation times and to study a possible association of these BSA-antibodies with autoantibodies (AAb) frequently detected in type 1 diabetic patients. For the overnight incubation time, there was an enormous overlap in the [I-125]BSA binding by serum samples between 52 newly diagnosed type 1 diabetic patients (mean [I-125]BSA binding 23.6 +/- 17.4%) and 54 healthy blood donors (mean [I-125]BSA binding 10.2 +/- 15.7%). By an incubation time of only 3 min the BSA-antibody prevalence was found to be 15.4% (8/52) for type 1 diabetic patients and 3.7% (2/54) for control subjects, However, there was no association between BSA-Ab and type 1 diabetes-associated antibodies as cytoplasmic islet cell antibodies (ICA), or glutamate decarboxylase autoantibodies. Our results confirm that (i) BSA-Ab occur more frequently in newly diagnosed type 1 diabetic patients compared with a healthy control group and (ii) that the BSA-Ab detected by the fluid-phase radioimmunoassay with an incubation time of 3 min are more disease-associated than the [I-125]BSA binding after an overnight incubation, The competitive BSA-Ab fluid-phase radioimmunoassay described is a simple and rapid method to detect antibodies specifically reactive with BSA. It is suggested that the humoral immune reactivity to BSA in type 1 diabetic patients probably reflects an unspecific defect of the immune system and gives no additionally diagnostic value about the type 1 diabetes.