Autoantibodies to glutamic acid decarboxylase in three patients with cerebellar ataxia, late-onset insulin-dependent diabetes mellitus, and polyendocrine autoimmunity

BACKGROUND: Glutamic acid decarboxylase (GAD) is the main target of humoral autoimmunity in stiff-man syndrome (SMS) and insulin-dependent diabetes mellitus (IDDM). GAD autoantibodies (GAD-Abs) are reported in a few patients with cerebellar ataxia, but their relevance is unclear. We describe three patients with cerebellar ataxia and GAD-Abs. METHODS: GAD-Abs were assayed by radioimmunoassay (RIA) and immunohistochemistry and confirmed by immunoblot of recombinant human GAD65. The GAD-Ab levels of the three patients with cerebellar ataxia were compared with those of five with SMS, 49 with IDDM, 64 with cerebellar ataxia of probable degenerative origin without associated autoimmune features, 14 non-IDDM islet cell antibody-positive first-degree relatives of IDDM patients, and 91 normal subjects. RESULTS: The three patients with ataxia and GAD-Abs were women (mean age, 63 years) with an isolated progressive cerebellar disorder, family history of IDDM, late-onset IDDM, and several positive serum organ-specific autoantibodies. Two patients had autoimmune thyroiditis, and one had pernicious anemia. CSF analysis demonstrated oligoclonal IgG bands and intrathecal synthesis of GAD-Abs. By RIA, GAD-Ab titers from the three patients were similar to those of SMS and significantly higher, without overlap, than the titers of IDDM patients. GAD-Abs were absent in the 64 patients with cerebellar ataxia and no evidence of autoimmune disorders. CONCLUSIONS: These findings suggest a link of GAD autoimmunity not only with SMS but also with cerebellar dysfunction. GAD-Abs should be sought in patients with cerebellar ataxia who have late-onset IDDM and other organ-specific autoimmune manifestations.     

High prevalence of antibodies to glutamic acid decarboxylase in comparison to islet cell antibodies in patients with long-standing insulin-dependent diabetes mellitus

To elucidate the clinical significance of antibodies to glutamic acid decarboxylase (GAD Ab) compared to islet cell antibodies (ICA) in recent-onset and long-standing insulin-dependent diabetes mellitus (IDDM). We examined GAD Ab and ICA in 29 recent-onset and 85 long-standing patients with IDDM. GAD Ab was detected by a radioimmunoassay kit using purified pig brain GAD as an antigen. The prevalence of GAD Ab in the recent-onset diabetic patients was 55.2%, slightly lower than that of ICA (65.5%). In contrast, the prevalence of GAD Ab in long-standing diabetic patients was 42.4%, which was significantly higher than that of ICA (23.5%) (p < 0.01). GAD Ab were consistently detected in approximately 40% of patients with long-standing disease, while ICA decreased according to duration of disease. The GAD Ab titer in ICA-positive patients (mean +/- SD, 1588.2 +/- 6755.1; range, 6-38574) was significantly higher than that in ICA-negative patients (mean +/- SD, 13.4 +/- 17.9; and range, 5-72 units) (p < 0.001). These findings suggest that GAD Ab are more useful than ICA to know participation of immune disorders in long-standing patients with IDDM.     

Peripheral osteopenia in adult patients with insulin-dependent diabetes mellitus

Alterations in bone metabolism in diabetes mellitus is a topic of special interest. Bone blood flow is increased in the distal limb of diabetic patients, which is believed to increase osteoclastic activity. We measure bone mineral density using dual-photon absorptiometry in the distal lower limb, the femoral neck, and the lumbar spine in 41 IDDM patients and in 30 control persons. In the diabetic group there was a 10% reduction of bone mineral density in the femoral neck (p < 0.01) and a 12% reduction in the distal limb (p < 0.001) compared with the control group. No significant difference was found in the lumbar spine (p = 0.22). Our data yield incidence for peripheral osteopenia in IDDM-patients, independent of any systemic bone disease such as osteoporosis. A link between decreased bone mineral density and diabetic neuropathy has been observed for the femoral neck (p < 0.001), but not for the distal limb or axial skeleton. Whether there is a common aetiological link or a casual connection between diabetic neuropathy and bone mineral density has still to be determined.     

Pancreatic beta cell function and antibodies to glutamic acid decarboxylase (anti-GAD) in Chinese patients with clinical diagnosis of insulin-dependent diabetes mellitus

Antibodies to glutamic acid decarboxylase (anti-GAD) and pancreatic beta cell secretory function were measured in 39 consecutive Chinese patients with a clinical diagnosis of insulin-dependent diabetes mellitus (IDDM) (19 males, mean +/- SD age. 37 +/- 15 years; body mass index (BMI), 22 +/- 4 kg/m2; mean duration of disease, 6.7 +/- 5.6 years). IDDM was defined on the basis of acute symptoms with heavy ketonuria (> 3+) or ketoacidosis at diagnosis, or requirement for continuous insulin treatment within one year of diagnosis. Insulin deficiency was defined as a post-glucagon stimulated plasma C-peptide concentration < or = 0.6 nmol/l. Overall, anti-GAD antibodies were positive (> 18 units) in 23% (n = 9) of these patients. Of the 39 patients, 29 (74%) were insulin deficient and 10 (26%) were non-insulin deficient. Anti-GAD antibodies were positive in 31% of the insulin-deficient patients but in none of the non-insulin-deficient group. Insulin deficiency and anti-GAD positivity were associated with younger age, earlier age of clinical onset and lower BMI. There were independent negative relationships between levels of anti-GAD antibodies and blood pressure and a positive relationship between insulin dosage and albuminuria. This study emphasises the difficulty in differentiating clinically between IDDM and NIDDM in Chinese patients. Despite the acute presentation, these patients had variable pancreatic beta cell secretory function. The varying duration of disease may partly explain the low prevalence of positive anti-GAD antibodies in these patients, but seems unlikely to explain fully the difference from Caucasian IDDM patients.     

Insulin resistance precedes microalbuminuria in patients with insulin-dependent diabetes mellitus

BACKGROUND: Insulin resistance has been associated with hypertension and with renal complications in patients with type 1 diabetes mellitus. Causal relationships have not been fully explained. METHODS: We investigated whether insulin resistance precedes microalbuminuria by measuring insulin resistance with a euglycaemic clamp in combination with indirect calorimetry in 16 uncomplicated type 1 diabetic patients and in six healthy control subjects. The patients had over 10 year duration of diabetes, and were expected to experience either a complication-free or complicated disease course within the next few years. They have thereafter been followed for the development of microalbuminuria for 3 years. RESULTS: In a euglycaemic insulin clamp glucose disposal was lower in diabetic patients compared with control subjects (7.5 +/- 2.9 and 12.6 +/- 2.0 mg/kg LBM/min; P<0.002), mainly due to impaired glucose storage (4.3 +/- 2.3 vs 8.6 +/- 1.6 mg/kg LBM/min; P<0.001). Three years later seven IDDM patients had albumin excretion rate over 30 mg/24 h; glucose disposal (5.5 +/- 2.1 vs 9.0 +/- 2.2 mg/kg LBM/min; P<0.01) had been lower in patients who developed microalbuminuria compared with those who remained normoalbuminuric. CONCLUSIONS: Insulin resistance predicts the increment in urinary albumin excretion. Insulin resistance depends mainly on impaired glucose storage in uncomplicated IDDM.     

Glycemic control and cardiopulmonary function in patients with insulin-dependent diabetes mellitus

BACKGROUND: We studied cardiopulmonary function during exercise in young subjects with long-standing insulin-dependent diabetes mellitus (IDDM) who have no clinical cardiopulmonary disease to determine the relationships of aerobic capacity, gas exchange, ventilatory power requirement, and cardiac output to chronic glycemic control. METHODS: Eighteen subjects with IDDM and 14 normal control subjects were studied. Nine diabetic subjects received twice daily insulin injections and had chronically elevated levels of glycosylated hemoglobin (hyperglycemic group); 9 other diabetic subjects received insulin via continuous infusion pumps and maintained chronic near-normal levels of glycosylated hemoglobin (normoglycemic group). At the end of at least 7 years of regular follow-up, aerobic capacity was determined by cycle ergometry. Lung volume, diffusing capacity, and cardiac output during exercise were measured by a rebreathing technique. Ventilatory power was measured by the esophageal balloon technique. RESULTS: Maximal work load and oxygen uptake were markedly impaired in chronically hyperglycemic diabetic patients associated with significant restrictions of lung volume, lung diffusing capacity, and stroke index during exercise. Membrane diffusing capacity was significantly reduced at a given cardiac index. The normoglycemic patients consistently showed less impairment than the hyperglycemic patients. CONCLUSION: Physiologically significant cardiopulmonary dysfunction develops in asymptomatic patients with long-standing IDDM. Chronic maintenance of near-normoglycemia is associated with improved cardiopulmonary function.     

Oligoclonal CD4+ CD57+ T-cell expansions contribute to the imbalanced T-cell receptor repertoire of rheumatoid arthritis patients

A peculiar feature of rheumatoid arthritis patients is that they carry clonally expanded CD4+ and CD8+ cells in the peripheral blood. While the distortion of the repertoire of CD8+ cells has been ascribed to the increase of CD8+ CD57+ large granular lymphocytes, often detected in these patients, the mechanism responsible for the clonal expansion of CD4+ cells remains unexplained. Here, we report that CD4+ CD57+ cells, that in healthy individuals represent a small subset of peripheral CD4+ lymphocytes, are significantly expanded in the peripheral blood of a considerable percentage of rheumatoid arthritis patients. Furthermore, the expansion of these lymphocytes appears to correlate with the presence of rheumatoid factor. The molecular analysis of the T-cell receptor variable beta segments expressed by the CD4+ CD57+ cells enriched in rheumatoid arthritis patients showed that they use restricted repertoires, that partially overlap with those of their CD4- CD57+ counterpart. The structural feature of the receptor ligand expressed by these cells revealed that their expansion is most likely mediated by strong antigenic pressures. However, since we also found that CD4+ CD57+ and CD4- CD57+ cells can share the same clonal specificity, it is likely that their selection is not mediated by conventional major histocompatibility complex restricted mechanisms. Thus, while our data demonstrate that CD4+ CD57+ cells play an important role in establishing the imbalance of the CD4+ cell repertoire observed in rheumatoid arthritis patients, they also suggest that these cells have common features with mouse CD4+ CD8- NK1.1+/T cells.     

Radioimmunoassay detects the frequent occurrence of autoantibodies to the Mr 65,000 isoform of glutamic acid decarboxylase in Japanese insulin-dependent diabetes

Glutamic acid decarboxylase antibodies (GAD65Ab) are common in new onset Caucasian insulin-dependent diabetic (IDDM) patients but it is unclear if this marker is also prevalent in patients of other ethnic backgrounds. We determined antibodies against human recombinant GAD in Japanese diabetic patients using a radioimmunoassay with competition between in vitro translated 35S-GAD65 and non-labelled recombinant human GAD65 (rhGAD65). GAD67 antibodies (GAD67Ab) were similarly analyzed but without antigen competition. In 73 Japanese diabetic patients, GAD65Ab were found in 11/16 (69%) of patients with short-duration (less than 5 yrs) IDDM, 6/23 (26%) with long-duration (5 or more yrs) IDDM and 10/20 (50%) with slowly progressive diabetes. High GAD65Ab levels were associated with concomitant autoimmune diseases (p = 0.021). GAD67Ab were found in 4/16 (25%) of patients with short-duration IDDM, 3/23 (13%) with long-duration IDDM and 2/20 (10%) with slowly progressive diabetes. In 14 non-insulin dependent diabetic (NIDDM) patients, GAD65Ab and GAD67Ab were not found (0/14) and 1/50 (2%) healthy controls were positive in either assay. Among the GAD67Ab-positive samples, 8/9 (88%) were also high level GAD65Ab positive, 7/9 (77%) were displaced by an excess of rhGAD65 and the antibody levels correlated (r2 = 0.573; p = 0.003). Our data are consistent with a strong association of GAD65Ab also in Japanese IDDM, and suggest that, when present, GAD67Ab are frequently directed to epitope(s) common to GAD65 and GAD67.     

Parasympathetic neuropathy associated with left ventricular diastolic dysfunction in patients with insulin-dependent diabetes mellitus

BACKGROUND: As extracorporeal shock wave lithotripsy (ESWL) is frequently carried out on an outpatient basis, it is crucial to choose an adequate analgesic with less adverse effect. This study evaluated the use of three different intravenous agents: fentanyl, tramadol HCl and tenoxicam in ESWL. METHODS: One hundred and twenty patients undergoing lithotripsy were randomly assigned to receive either intravenous fentanyl 1 microgram/kg, tramadol HCl 1.5 mg/kg or tenoxicam 0.3 mg/kg before lithotripsy. Pain intensity was recorded using verbal rating pain scales (VRPS). Cases without adequate analgesia (VRPS > 4) or could not tolerate the pain, additional bolus of fentanyl 25 micrograms were given until adequate analgesia was achieved. Side effects were recorded as well. RESULTS: No significant differences were found among groups in demographic data, VRPS, number of total shock waves, cases with supplementary fentanyl, mean dose of supplementary fentanyl or the incidence of dizziness. However, the incidence of nausea or vomiting was significantly higher in fentanyl and tramadol groups comparing with tenoxicam group (15.0% and 25.0% vs. 0.0%). Oxygen saturation in fentanyl group was also significantly lower than the other two groups (p < 0.01). In addition, VRPS had a significant correlation with voltage intensities (p < 0.05). CONCLUSIONS: Lithotripsy can be satisfactorily performed by employing fentanyl, tramadol or tenoxicam for analgesia; tenoxicam apparently offers a better analgesic quality with less side effect. Furthermore, the need for stronger analgesia during larger voltage intensity should be tailored to the needs of the individuals.     

Characterization of ovomucoid-specific T-cell lines and clones from egg-allergic subjects

Three-dimensional gel culture systems represent conditions that mimic the differentiated state of mesenchymal cells in vivo. We examined gel contraction, cell growth, and phenotypic modulation of rabbit arterial SMC in three-dimensional gel culture. The gel contraction rate was dependent on the collagen type; that is, the contraction by freshly isolated SMC was faster and more pronounced in type I collagen than in type III collagen. In contrast, the phenotypic modulation of SMC was independent of collagen type. The major portion of cells in both type I and III collagens with growth factors underwent transition from a contractile (G0 phase) to a synthetic phenotype (G1B phase), but this transition was clearly delayed compared with that on collagens. The cells had hardly begun DNA synthesis in either collagen type and failed to proliferate even after 10 days of culture. These results indicate that collagen type is important in gel contraction by vascular SMC, while the organization of collagen fibrils (two-dimensional vs three-dimensional) is more critical in the phenotypic transition and proliferation of these cells. However, the more specific organization of extracellular matrix than the collagen gel culture system may be necessary to maintain the contractile phenotype of SMC.     

Characteristics and maintenance of CD8+ T-cell clones found in old mice

Old mice, like old human beings, contain large clones of CD8+ T-cells. These cells grow poorly in tissue culture, therefore it is difficult to maintain the cells in vitro. The cells can be grown after transfer to sublethally irradiated mice. This technique will be useful in further studies on the properties of the cells. Based on observations from such transfer experiments, we conclude that: (1) expansion of the T-cell clones in recipients is dramatic but slow; (2) chance events caused by endogenous antigens or gene mutations rather than exogenous antigens may account for the expansion of these clones; and (3) the expanded T-cell clones are benign and do not cause malignancies.     

Biological activity of C-peptide on the skin microcirculation in patients with insulin-dependent diabetes mellitus

19 insulin-dependent diabetes mellitus (IDDM) patients participated in a randomized double-blind crossover investigation to investigate the impact of human C-peptide on skin microvascular blood flow. The investigation was also carried out with 10 healthy volunteers. Blood pressure, heart rate, blood sugar, and C-peptide levels were monitored during a 60-min intravenous infusion period of C-peptide (8 pmol kg-1 min-1) or saline solution (154 mmol liter-1 NaCl), and 30 min after stopping the infusion. During the same time period, capillary blood cell velocity (CBV), laser Doppler flux (LDF), and skin temperature were assessed in the feet. In the verum arm, C-peptide levels increased after starting infusion to reach a maximum of 2.3+/-0.2 nmol liter-1 after 45 min, but remained below 0. 15 nmol liter-1 during the saline treatment. Baseline CBV was lower in diabetic patients compared with healthy subjects (147+/-3.6 vs. 162+/-4.2 micron s-1; P < 0.01). During C-peptide administration, CBV in IDDM patients increased progressively from 147+/-3.6 to 167+/-3.7 micron s-1; P < 0.001), whereas no significant change occurred during saline infusion or in healthy subjects. In contrast to the CBV measurements, the investigation of LDF, skin temperature, blood pressure, heart rate, or blood sugar did not demonstrate any significant change during the study. Replacement of human C-peptide in IDDM patients leads to a redistribution in skin microvascular blood flow levels comparable to levels in healthy subjects by increasing the nutritive CBV relative to subpapillary arteriovenous shunt flow.     

Prevalence and magnitude of osteopenia associated with insulin-dependent diabetes mellitus

Effects of bilateral chemical inactivation of the nucleus tractus solitarius (NTS) by microinjection of kainic acid (KA) on fluid and NaCl absorption across the jejunum were examined in anesthetized Sprague-Dawley rats. Jejunal fluid and NaCl absorption was measured in a jejunal loop before and after the microinjection of artificial cerebrospinal fluid (aCSF) or KA into the NTS. Net fluid and NaCl absorption was not altered by a microinjection of aCSF. However, net fluid (from 1.12 +/- 0.07 to 1.66 +/- 0.06 ml/30 min) and NaCl (Na+ from 164.5 +/- 10.1 to 243.3 +/- 7.1 muEq/30 min; and Cl- from 175.9 +/- 7.5 to 260.8 +/- 6.5 muEq/30 min) absorption was significantly increased by the chemical inactivation of the NTS. To examine the efferent mechanism of the increased net absorption induced by the NTS inactivation, mesenteric nerve activity (MNA) was measured before and after the inactivation of the NTS. MNA was significantly increased by 165.9 +/- 74.2% after the bilateral inactivation of the NTS. Furthermore, absorption experiments were conducted in rats with pretreatment of atropine (acetylcholine-antagonist) or yohimbine (specific alpha 2-antagonist). In atropine treated rats, net jejunal absorption was significantly increased by the inactivation of the NTS. However, the increase in net absorption induced by the inactivation of the NTS was completely abolished by pretreatment with yohimbine. These results suggest that the NTS has a tonic suppression on jejunal absorption through alpha 2-adrenergic mechanism.     

The serum growth hormone-binding protein is reduced in young patients with insulin-dependent diabetes mellitus

Despite elevated serum concentrations of GH, longitudinal growth is stunted in a considerable number of children and adolescents with insulin-dependent diabetes mellitus (IDDM). To elucidate, whether reduced peripheral action of GH contributes to this phenomenon, GH-binding protein (GH-BP) activity was measured in 117 children and adolescents with IDDM (mean age 14.6 yr, range 4.5-28 yr) and 132 healthy controls (13.1 yr, 6.3-26 yr). Serum was incubated with 125I-GH, then chromatographed on a Sephacryl S200 column (1.8.100 cm), apparent binding of 125I-GH to GH-BP was corrected for the amount of endogenous GH present in the sample. GH-BP activity was significantly lower in IDDM patients, with a corrected binding of 16.8 +/- 0.6% compared to 21.3 +/- 0.7% in control children (mean +/- SE; P < 0.0001, Wilcoxon-test). Previous studies demonstrated that GH-BP is increased in healthy overweight children. In contrast, in IDDM children, GH-BP was reduced despite a moderate degree of overweight (z-score for weight: +0.94 +/- 0.12; mean +/- SE). Reduced serum GH-BP activity in IDDM children is further accentuated when compared to healthy children with a similar degree of overweight (22.8 +/- 0.5%; n = 44). Based on this novel finding, we conclude that decreased GH receptor density may explain reduced growth velocity despite increased secretion of GH in some IDDM children.     

Differential susceptibility to HIV-GP120-sensitized apoptosis in CD4+ T-cell clones with different T-helper phenotypes: role of CD95/CD95L interactions

The susceptibility of Th1 and Th2 cell clones to apoptosis following HIV-gp120/CD4 cross-linking and TCR activation was investigated. We show that only Th1 clones are susceptible to HIV-gp120-sensitized apoptosis, although both types of clones express similar levels of CD4 and bind similar amounts of recombinant gp120. Both types of clones, however, undergo apoptosis induced by CD95 cross-linking with agonistic monoclonal antibody (MoAb). Apoptosis induced by gp120 in the Th1 clones is inhibited by either an anti-CD95 neutralizing MoAb or an anti-CD95L neutralizing MoAb as well as by a specific interleukin-1 beta converting enzyme (ICE) inhibitor. When triggered to apoptosis by gp120, Th1 but not Th2 clones express both cell-associated and soluble CD95L. The CD95L produced by Th1 clones induces cell death, inhibitable by anti-CD95 neutralizing MoAb, of CD95 positive Jurkat cells. These data suggest that, like activation-induced apoptosis, HIV-gp120 sensitized apoptosis in Th1 clones occurs via CD95/CD95L interaction and that lack or insufficient production of CD95L is responsible, at least in part, for the resistance of Th2 clones to such apoptosis.     

High prevalence of celiac disease among patients with insulin-dependent (type I) diabetes mellitus

OBJECTIVES: Diagnosis of unrecognized celiac disease is potentially important. The prevalence of celiac disease in patients with insulin-dependent diabetes mellitus is uncertain. We report the prevalence of celiac disease in a stratified random sample (n = 101) of adult insulin-dependent diabetic patients (age, 18-59 yr) attending our clinic, and in an age- and sex-matched control group (n = 51). METHODS: Screening was by anti-endomysial antibody, measured by indirect immunofluorescence using sections of human umbilical cord. RESULTS: Celiac disease had not been suspected in any patient at the time of screening. Eight patients tested positive for anti-endomysial antibody, all of whom had a distal duodenal biopsy performed. Five patients had histologic evidence of celiac disease. One patient with negative histology was receiving immunosuppressive therapy for a renal-pancreas transplant. Of the five patients with abnormal histology, two improved on gluten restriction, one was unable to comply, one refused treatment, and one was lost to follow-up. No control subject tested positive for endomysial antibody. CONCLUSIONS: Patients with insulin-dependent diabetes have an increased prevalence of celiac disease. Because most cases are clinically unrecognized, consideration should be given to screening all insulin-dependent diabetes mellitus patients with endomysial antibodies.