Pre-meal insulin aspart compared with pre-meal soluble human insulin in type 1 diabetes.

Insulin aspart has been shown, in medium-term studies, to achieve reductions in HbA(1c) without increasing the risk of major hypoglycaemia compared with pre-meal human insulin. The aim of the present 3-year study was to evaluate the long-term safety and efficacy of insulin aspart in people with type 1 diabetes. This was a 30-month extension of a multinational, multicentre, open-label, parallel-group study of 753 people with type 1 diabetes, originally randomly allocated to treatment with insulin aspart or unmodified human insulin before meals, with NPH insulin as basal insulin. Main outcomes measures were hypoglycaemia (major or minor), adverse events and HbA(1c). As insulin aspart became commercially available in some countries before the end of the trial, analyses of HbA(1c) used 30-month data to maintain statistical power. The relative risk estimate of major hypoglycaemia was similar between treatment groups (relative risk [RR] 1.00 [95% CI 0.72, 1.39]). The risk of having a minor hypoglycaemic episode was higher with insulin aspart than with human soluble insulin (RR 1.24 [1.09, 1.39] p=0.024). Insulin aspart was significantly superior to human insulin with respect to overall glycaemic control, with a baseline-adjusted HbA(1c) difference of -0.16 (-0.32, -0.01)% (p=0.035). Insulin aspart was well tolerated and effective during long-term treatment. The HbA(1c) advantage was maintained with insulin aspart without any adverse impact on the rate of major hypoglycaemia.

Transplantation of human fetal pancreas: experience in thymusaplastic mice and rats and in a diabetic patient.

The aim of the present study was to investigate if thymusaplastic nude mice and rats are favorable as recipients for transplantation of human fetal pancreas. Twenty human fetal pancreases were transplanted subcutaneously to 20 nude mice, and six human fetal pancreases were transplanted to six rats. The xenografts showed histotypical development of islets of Langerhans. Insulin, glucagon, somatostatin, and pancreatic polypeptide immunoreactivities were also seen in very early stages of the transplant development within the monolayered ducts. With the described "epigastric pouch technique" in rats, we co-ld demonstrate a new in vivo method for selective stimulations and simultaneous blood sampling from tissue-isolated xenografts. Transplantation of human fetal pancreas to the brachioradial muscle of an insulin-dependent patient in combination with a kidney transplant revealed that rejection crises of the kidney led to necrosis of the pancreas transplant, whereas rejection of the kidney was overcome by steroid pulse therapy.

IDDM patients neither show humoral reactivities against endogenous retroviral envelope protein nor do they differ in retroviral mRNA expression from healthy relatives or normal individuals.

Recently, human endogenous retrovirus type K (HERV-K [IDDMK(1,2)22]) was isolated from an IDDM patient's beta-cell supernatant and shown to be implicated in expression as a superantigen. Furthermore, HERV-K RNA was found in plasma samples from newly diagnosed patients but not in those from healthy control subjects. We had earlier identified the presence of a HERV-K long terminal repeat element of the HLA DQ gene (DQ-LTR) to be positively associated with IDDM, which led us to investigate whether DQ-LTR is related to transcription of the putative retroviral superantigen. Additionally, we sought immunological evidence to determine whether those retroviral antigens could evoke an antibody response. Patients with IDDM (n = 14), Hashimoto's thyroiditits (n = 5), and Graves' disease (n = 12), as well as healthy control subjects (n = 12), were investigated, as were four nuclear families of Graves' disease patients and two of IDDM patients. RNA was isolated from plasma and peripheral blood lymphocytes and subjected to reverse transcription-polymerase chain reaction for transcripts of the env region of the HERV-K (IDDMK(1,2)22) sequence. We identified env transcripts in both plasma and peripheral blood lymphocytes in all individuals studied: patients with recent-onset or long-standing IDDM, their relatives, and healthy control subjects, as well as patients with thyroid autoimmune disorders. Furthermore, we screened the sera of patients (n = 62) and control subjects (n = 35) for evidence of humoral immunity against HERV-K by Western blot specific for the ENV protein. Similar frequencies of antibody-positives were observed both in patients with IDDM (29%) and in healthy control subjects (26%). We conclude that neither the ubiquitous HERV-K transcripts nor the comparable percentage of ENV protein antibodies are associated with IDDM. An earlier, presymptomatic antibody response against HERV-K (IDDMK(1,22)22) ENV cannot be ruled out. However, the superantigen hypothesis of an endogenous retrovirus in beta-cell autoimmunity awaits confirmation.

Vitamin D receptor allele combinations influence genetic susceptibility to type 1 diabetes in Germans.

Vitamin D has been shown to exert manifold immunomodulatory effects. Because type 1 diabetes is regarded to be immune-mediated and vitamin D prevents the development of diabetes in the NOD mouse, we investigated the role of the vitamin D receptor (VDR) gene as a candidate for type 1 diabetes susceptibility. A total of 152 Caucasian families with at least one affected offspring were genotyped for four VDR restriction-site polymorphisms (FokI, BsmI, ApaI, and TaqI). Whereas the BsmI, ApaI, and TaqI polymorphisms are in strong linkage disequilibrium with each other, no significant linkage disequilibrium with the FokI site was observed. Extended transmission disequilibrium testing (ETDT) was used to detect preferential transmission of allelic combinations to affected offspring. We found significant haplotype-wise ETDT results for the BsmI/ApaI/TaqI (chi2 = 18.886, df = 7, P = 0.0086), the BsmI/TaqI (chi2 = 8.373, df = 3, P = 0.0389), and theApaI/TaqI (chi2 = 17.182, df = 3, P = 0.0006) haplotypes. The "At" and "Bt" alleles confer an increased risk, whereas "AT" and "at" are protective. The combination with the strongest susceptibility was the "BAt" haplotype (64% transmitted, P = 0.0106). Analysis of the FokI site does not provide more information on susceptibility (FokI/BsmI/ApaI/TaqI [chi2 = 24.702, df = 15, P = 0.0541]). These findings suggest a linkage of VDR itself or a nearby gene with type 1 diabetes susceptibility in Germans, confirming respective observations previously made in Indian Asians.

Isolation of thyroid cells obtained by fine-needle aspiration biopsy.

Usually thyroid cells isolated from tissue obtained by surgery or thyroid cell lines are used to investigate the pathogenesis of autoimmune thyroid diseases. Isolation and cultivation of thyrocytes from fine-needle aspiration biopsy (FNAB) has not yet been published. The aim of this study was to isolate and cultivate thyrocytes from samples of FNAB. FNAB samples were obtained from nine adults and nine children with Hashimoto's thyroiditis (HT). The aspiration material was filtered resulting in small samples of tissue on the surface of the filter membrane. These tissue fragments were digested by collagenase I and dispase II. The yielding cells were cultivated for 3 weeks in Ham's F12 Kaighn's Modification medium in presence of 1 mU/mL bovine thyrotropin (TSH), 10 microg/mL human insulin, 6 microg/mL transferrin, and 10(-8) M hydrocortisone. Finally, isolated thyroid cells were characterized by determination of gene expression of thyrotropin receptor (TSHR), thyroperoxidase (TPO), and thyroglobulin (Tg) using a nested reverse transcriptase-polymerase chain reaction (RT-PCR). Thyroid cells obtained by FNAB can be maintained over a time period of approximately 3 weeks. Depending on the sample size a final number of 1000-14,000 cells was gained per FNAB. In addition, all cells isolated by the described method expressed TPO mRNA. TSHR mRNA was found in 4 samples, whereas 15 samples were Tg mRNA-positive. There were no differences with respect to the expression TSHR and TPO mRNA between samples from adults and children. The isolation and cultivation of thyroid cells obtained by FNAB has been established. In contrast to surgical specimen, this technique provides an easy access to thyrocytes derived from individual patients allowing repeated sampling to investigate the time progression of the chronic disease or the effect of treatment over time.

Graves' disease: xenotransplantation model (athymic nude mice).

The immunological mechanisms leading to Graves' disease are not yet fully understood. The athymic nude mouse has immunological properties which allow in vivo studies concerning autoimmune thyroid diseases with special regard to the interaction of TSH, TSH receptor antibodies, cytokines, antithyroid drugs, TSH receptor antagonists and human lymphocytes. In our own studies thyroid tissues of patients with Graves' disease, toxic adenomas and non-toxic nodular goiter were xenotransplanted to athymic nude mice. Histology, morphology and function of the transplants were examined 2 days to 2 weeks after injection of bovine TSH, interferon-gamma, Graves' sera with or without addition of a TSH-receptor antagonist and lymphocytes of patients with Graves' disease. Thyroid transplants can be stimulated by TSH, interferon-gamma, Graves' sera and immunoglobulin G. Additional treatment with asialoagalacto-hCG inhibits stimulation of the immunoglobulin. Furthermore, preliminary results show, that engrafted peripheral and especially intrathyroidal lymphocytes from patients with Graves' disease specifically migrate into human thyroid transplants ("homing") and are able to induce functional and histological changes in these tissues. In summary, the xenotransplantation model is well suited for studies concerning pathogenesis, diagnosis and therapy of autoimmune thyroid diseases.

Bone mineral density changes and bone turnover in thyroid carcinoma patients treated with supraphysiologic doses of thyroxine.

The aim of this one-year prospective study was to determine whether longterm thyroxine treatment is a risk factor for elevated bone turnover, loss of bone mass and subsequent development of osteoporosis. Premenopausal women (N = 19), and men (N = 9) suffering from differentiated thyroid gland carcinoma in the mean age of 39.0 +/- 8.0 years and 41.8 +/- 10.0 years were investigated. All of them had undergone a total thyroidectomy and subsequent thyroxine therapy. The duration of the TSH-suppressive therapy prior to the the beginning of our study was 9.4 +/- 6.4 years in the female and 8.1 +/- 6.0 years in the male group. The prospective observation was performed by dual X-ray absorptiometry (DXA) at the spine and the femoral neck and by single-photon absorptiometry (SPA) at the distal radius. Laboratory testings included thyroid hormones T3, T4 and TSH, serum calcium, phosphate and PTH, and urinary calcium and phosphate from spontaneous and 24-hour urine samples. Markers of bone formation (osteocalcin, alkaline phosphatase and PICP) and resorption (Ca/Cr and ICTP) were determined. Statistically significant loss of bone mass was observed only on the distal radius in males (p<0.05). At the lumbar spine and femoral neck, only a minor bone loss was registered in a small number of patients. Almost 50 % of the females showed values above the reference range. In more than 30 % of the females, and smaller number of male patients, ICTP values ranged above the reference range, corresponding to elevated bone turnover. These two variables exhibited a slight correlation with bone density at the measured skeletal areas, mostly considering the male group. The results are a proof that accelerated bone turnover and subsequent bone loss occurs during TSH-suppressive thyroxine therapy. In future prospective studies a prolonged time of observation will be necessary, as well as to increase the number of studied patients, in order to better assess the relative risk of osteoporosis in patients undergoing TSH-suppressive treatment more precisely.

alpha-Lipoic acid treatment decreases serum lactate and pyruvate concentrations and improves glucose effectiveness in lean and obese patients with type 2 diabetes.

OBJECTIVE: We examined the effect of lipoic acid (LA), a cofactor of the pyruvate dehydrogenase complex (PDH), on insulin sensitivity (SI) and glucose effectiveness (SG) and on serum lactate and pyruvate levels after oral glucose tolerance tests (OGTTs) and modified frequently sampled intravenous glucose tolerance tests (FSIGTTs) in lean (n = 10) and obese (n = 10) patients with type 2 diabetes. RESEARCH DESIGN AND METHODS: FSIGTT data were analyzed by minimal modeling technique to determine SI and SG before and after oral treatment (600 mg, twice a day, for 4 weeks). Serum lactate and pyruvate levels of diabetic patients after glucose loading were compared with those of lean (n = 10) and obese (n = 10) healthy control subjects in which SI and SG were also determined from FSIGTT data. RESULTS: Fasting lactate and pyruvate levels were significantly increased in patients with type 2 diabetes. These metabolites did not exceed elevated fasting concentrations after glucose loading in lean patients with type 2 diabetes. However, a twofold increase of lactate and pyruvate levels was measured in obese diabetic patients. LA treatment was associated with increased SG in both diabetic groups (lean 1.28 +/- 0.14 to 1.93 +/- 0.13; obese 1.07 +/- 0.11 to 1.53 +/- 0.08 x 10(-2) min-1, P < 0.05). Higher SI and lower fasting glucose were measured in lean diabetic patients only (P < 0.05). Lactate and pyruvate before and after glucose loading were approximately 45% lower in lean and obese diabetic patients after LA treatment. CONCLUSIONS: Treatment of lean and obese diabetic patients with LA prevents hyperglycemia-induced increments of serum lactate and pyruvate levels and increases SG.

Chronic selective hypertriglyceridemia impairs endothelium-dependent vasodilatation in rats.

OBJECTIVE/METHODS: In order to investigate whether selective hypertriglyceridemia impairs endothelium-dependent vasodilatation in the rat hindlimb, rats were selectively bred to establish two strains, one with a pronounced hypertriglyceridemia (HT) and the other with normal plasma levels of triglycerides (LT). RESULTS: Carotid arteries and aortae removed from 3, 6, 9 and 12 month old LT- and HT-rats exhibited a normal morphology. However, marked morphological differences were observed between vessels from 18-20 month old HT- and LT-rats. The endothelium-dependent vasodilator acetylcholine (2 to 50 micrograms/kg), administered into the iliac artery, elicited a concentration-dependent increase in hindlimb blood flow which was not different in 3, 6 and 9 month old LT- or HT-rats but was impaired in 12 and 18-20 month old HT-rats. In contrast the endothelium-independent vasodilator sodium nitroprusside enhanced blood flow in both strains to a similar extent. Neither administration of the nitric oxide (NO) synthase (NOS) substrate, L-arginine, nor the NOS inhibitor NGnitro-L-arginine, affected the responsiveness to endothelium-dependent vasodilators in 12 month old HT-rats. These attenuated responses could not be attributed to a decrease in endothelial NOS expression as Western blot analysis revealed identical levels of this enzyme in the aortae and carotid arteries from LT- and HT-rats. Determination of superoxide anion (O2-) formation however, demonstrated a markedly elevated production of O2- in aortae from HT-rats. CONCLUSION: We conclude that chronic selective hypertriglyceridemia, an independent risk factor in the development and progression of atherosclerosis, leads to an endothelial dysfunction which is associated with an increased vascular O2- production and a subsequent decrease in bioavailable NO.

Heterotransplantation of human fetal pituitaries in nude mice.

The heterotransplantation of minced human fetal pituitaries into adult thymus-aplastic nude mice is described. Development and growth of such grafts were observed in 16 of 21 recipient mice. Histological examinations of the transplants showed typical adenohypophyseal cells. Neurohypophyseal cells could never be detected. The levels of human growth hormone (hGH) varied between 0.8 and 42.0 ng/ml in the plasma of the hosts (mean value 7.2 ng/ml, by radioimmunoassay).

Correlation of microsomal antibodies with the intensity of the intrathyroidal autoimmune process in Graves' disease.

Graves' disease is an organ-specific autoimmune disease, and intrathyroidal lymphocytes seem to be the major source of thyroid autoantibodies. Consequently, the intensity of the intrathyroidal lymphocytic infiltration is generally believed to reflect the activity of the autoimmune process. We, therefore, investigated the correlation of microsomal (enzyme immunoassay), thyroglobulin (RIA), and TSH receptor antibodies (RRA) with the degree of intrathyroidal infiltration by immunoglobulin G-producing plasma cells, activated T-cells, antigen-presenting cells, and the total number of lymphocytes. The immunocompetent cells were identified immunohistologically with monoclonal antibodies for immunoglobulins kappa and lambda, UCHL1, and the S100 antibody, respectively, in 26 thyroid glands of patients suffering from Graves' disease. The intensity of lymphocytic infiltration was determined by the point-counting method and by counting all lymphocytes and the labeled lymphocytes in 3 x 51 visual fields or 3 slides/thyroid gland. Microsomal antibodies correlated significantly (P = 0.001) with the total number of lymphocytes (r = 0.86), kappa (r = 0.71), lambda (r = 0.71), UCHL1 (r = 0.9), and S100 (r = 0.9) positive cells. These correlations were also significant for thyroglobulin antibodies. However, TSH receptor antibodies showed no significant correlations with any of the populations of immunocompetent cells. Patients with preoperatively undetectable TSH receptor or microsomal antibodies showed a broad variation of intrathyroidal infiltration by the immunocompetent cells investigated. Microsomal antibody titers, therefore, seem to reflect the intensity of the intrathyroidal autoimmune process in Graves' disease better than TSH receptor antibodies. However, the broad baseline variation in intrathyroidal infiltration observed with nondetectable thyroid antibodies will not always allow determination of the intensity of the intrathyroidal autoimmune process from microsomal or thyroglobulin antibody titers.

Lack of intrathyroidal tumor necrosis factor alpha in Graves' disease.

In vitro tumor necrosis factor alpha (TNF alpha) exerts a synergistic action on HLA class II expression and a cytotoxic action in FRTL-5 cells. Therefore, a role for TNF alpha as a local mediator of cell destruction in thyroid autoimmunity has been postulated. To elucidate the in vivo significance of these and other in vitro findings for the pathophysiology of Graves' disease we investigated 11 thyroid glands of patients suffering from Graves' disease for TNF alpha. In situ hybridization was done with a TNF alpha probe synthesized with T7 polymerase on a 750-base pair EcoRI fragment of the coding region. Primers at positions 152 and 854 in the TNF alpha copy DNA sequence were used for reverse polymerase chain reaction (PCR) amplification of TNF alpha in 5 patients. Immunohistological staining for TNF alpha was done with a mouse monoclonal antibody. We could not detect any TNF alpha and TNF alpha messenger RNA in thyroid tissue of 11 patients suffering mostly from relapsing Graves' disease by immunohistology and in situ hybridization as well as reverse PCR, respectively. A faint signal could be detected by reverse PCR in control thyroid tissue from a patient with recurrent goiter. This lack of intrathyroidal TNF alpha in relapsing Graves' disease is in accordance with a lack of increased TNF alpha production by T cell clones isolated from Graves' disease thyroid glands and contrasts with previous in vitro results. Since protective TNF actions have been demonstrated in other autoimmune diseases it could therefore be envisaged that the lack of intrathyroidal TNF alpha may be associated with the relapse of Graves' disease in our patients.

Methimazole has no dose-related effect on the intensity of the intrathyroidal autoimmune process in relapsing Graves' disease.

Declining thyroid autoantibodies during treatment and decreased lymphocytic infiltration after treatment of patients with Graves' disease suggest immunosuppressive actions of antithyroid drugs. However, the recent report of similar relapse rates after low and high dose carbimazole treatment of Graves' disease seems to contradict the immunosuppression thesis. We therefore determined the intrathyroidal methimazole concentrations with a high performance liquid chromatography method in 17 patients undergoing subtotal thyroid resection for relapsing Graves' disease. The intensity of the intrathyroidal infiltration by immunoglobulin G-producing plasma cells, activated T cells, and antigen presenting cells, and the total number of lymphocytes were identified immunohistologically with monoclonal antibodies for kappa- and lambda-immunoglobulin light chains, UCHL1, and the S100 antibody, respectively, followed by morphometry. The intrathyroidal methimazole concentration and the cumulative preoperative methimazole doses did not correlate with the intensity of the intrathyroidal infiltration by any of these immunocompetent cells. Comparison of groups with significantly different intrathyroidal methimazole concentrations (134 ng/g, n = 8 vs. 993 ng/g, n = 7) showed no significant differences for any of the intrathyroidal immunocompetent cells. These findings suggest that there is no dose-related effect of methimazole on the intensity of the intrathyroidal autoimmune process of patients with relapsing Graves' disease. They provide an explanation for why it does not seem justifiable to recommend higher methimazole doses than those required for the control of hyperthyroidism with the goal of immunosuppression.

The codon 17 polymorphism of the CTLA4 gene in type 2 diabetes mellitus.

Several studies have demonstrated an association of CTLA4 (IDDM12) alanine-17 with type 1 diabetes, but CTLA4 variants have not yet been investigated in type 2 diabetes. The CTLA4 exon 1 polymorphism (49 A/G) was analyzed in 300 Caucasian patients with type 2 diabetes and 466 healthy controls. All patients were negative for glutamate decarboxylase and islet cell antibodies. CTLA4 alleles were defined by PCR, single-strand conformational polymorphism, and restriction length fragment polymorphism analysis using BBV:I. The distribution of alleles as well as the genotypic and phenotypic frequencies were similar among patients and controls [AA, 42 vs. 39%; AG, 47 vs. 46%; GG, 11 vs. 15%, P = not significant (n.s.); A/G, 65/35% vs. 62/38%, P = n.s.; alanine/threonine 92/58% vs. 85/61%, P = n.s.]. However, detailed analysis of clinical and biochemical parameters revealed a tendency of GG (alanine/alanine) toward younger age at disease manifestation (46.8 +/- 0.8 vs. 49.5 +/- 0.8 yr, mean +/- SEM), lower body mass index (21.4 +/- 0.5 vs. 24.4 +/- 0.5 kg/m(2), P = 0.042), and basal C-peptide level (0.33 +/- 0.07 vs. 0.53 +/- 0.07nmol/L), as well as earlier start of insulin treatment (5.8 +/- 1.2 vs. 8.7 +/- 0.6 yr) and higher portion of patients on insulin (71 vs. 61%). Patients with the AA genotype were significantly less likely to develop microangiopathic lesions (P < 0.0005). No differences were found for hypertension or family history of type 2 diabetes. In conclusion, CTLA4 alanine-17 does not represent a major risk factor for type 2 diabetes. Additional studies on larger groups and different ethnic groups are warranted to clarify the association of the GG genotype with faster ss-cell failure and the lower rate of microvascular complications in AA carriers.

Susceptibility to thyroid autoimmune disease: molecular analysis of HLA-D region genes identifies new markers for goitrous Hashimoto's thyroiditis.

Hashimoto's thyroiditis has been shown to be associated with the HLA-specificities DR4 and DR5. Since former association studies yielded variable results, we used novel molecular typing methods to assess predisposing immunogenetic factors. Gene analysis of the HLA-DR-DQ and tumor necrosis factor region was performed in a group of Hashimoto's thyroiditis patients and randomly chosen controls using standards and nomenclature of the 10th International Histocompatibility Workshop. Genomic DNA of patients and controls was analyzed using a cDNA probe of the DQB1 gene. The resulting restriction fragment patterns allowed the determination of newly defined DQw-types 1-9. We find the strongest relative risk conferred by DQw7 (RR = 4.7), that is observed in 36 of 64 patients (56%) and only 21 of 98 controls (21%) (P corr less than 0.002). Comparison of DNA sequence variation in the DQB1 gene, that is found predominantly in Hashimoto's thyroiditis patients, indicates that codons 45 and 57 are critical features in DQw7 which distinguish it from other DQw specificities. The adjacent DQA1 genes also display a significant association with Hashimoto's thyroiditis (DQA1*0201/*0301 heterozygotes were found in 37% of patients and 15% controls, P less than 0.03). No significant association could be found with polymorphisms of the tumor necrosis factor gene. These results provide a new basis for the concept of genetic susceptibility in Hashimoto's thyroiditis and will help to elucidate the underlying autoimmune mechanisms that lead to disease at the functional level.

Susceptibility and resistance alleles of human leukocyte antigen (HLA) DQA1 and HLA DQB1 are shared in endocrine autoimmune disease.

Because particular human leukocyte antigen (HLA) DQ alleles are the major predisposing factors for type 1 diabetes mellitus (IDDM), we investigated whether they are shared by other endocrine autoimmune diseases. We, therefore, analyzed the HLA DQ genotypes of 171 patients with IDDM, 271 with Graves' disease (GD), 65 with Hashimoto's thyroiditis, 51 with postpartum thyroiditis, 53 with Addison's disease (AD), and 271 healthy controls. HLA DQA1 and DQB1 alleles were defined by polymerase chain reaction and sequence-specific oligonucleotide hybridization as well as by single strand conformational polymorphism analysis. HLA DQA1*0501 was significantly more frequent in IDDM (60%), GD (65%), and AD (70%) than in controls (43%); DQA1*0301 was significantly more frequent only in IDDM (67% vs. 30% controls). The heterozygous state DQA1*0301/*0501 was found in 9% of controls and 35% of IDDM (relative risk, 5.6). An arginine at position 52 on either DQA1 allele was significantly more frequent in patients with IDDM (94%), GD (80%), and AD (89%) compared with controls (66%). HLA DQB1*0201 and DQB1*0302 were more frequent in IDDM patients (*0201, 62% vs. 36% in controls, *0302, 59% vs. 19% controls), whereas DQB1*0602 was less frequent in IDDM (4%) and GD (18% vs. 31% of controls). In conclusion, endocrine autoimmunity has a common immunogenetic background; susceptibility is conferred by DQA1*0501 as well as an arginine at position 52 of DQA1 alleles, and protection against IDDM and GD is conferred by DQB1*0602.

Tumor necrosis factor beta gene polymorphisms in Graves' disease.

The physical mapping of tumor necrosis factor alpha (TNF alpha) and lymphotoxin (TNF beta) genes to the short arm of chromosome 6 in man between the loci for histocompatibility leucocyte antigens (HLA)-B and the complement system focused attention to this genetic region that controls immune responses in many ways. It also holds susceptibility genes for a variety of autoimmune disorders that are linked to specific alleles of loci in the HLA D subregion. We have recently identified a TNF restriction fragment length polymorphism with the enzyme NcoI (K. Badenhoop, G. Schwarz, J. Trowsdale, et al. Diabetologia. 1989;32:445-8). The less frequent fragment of 5.5 kilobase (kb) is in strong linkage disequilibrium with the HLA haplotype A1B8DR3. Since Graves' disease is linked to A1B8DR3, we analyzed TNF gene polymorphisms in a large group of Graves' disease patients and normal controls derived from four Centers. We show here a significant association of TNF beta polymorphisms with Graves' disease. The patients have less homozygotes for the 10.5 kb band (60 of 174, 34%) and more heterozygotes 10.5/5.5 kb (96 of 174, 55%), than 173 controls (49% homozygotes 10.5 kb and 42% heterozygotes; chi 2 = 7.45, P less than 0.03). When DR3+ patients and controls were analyzed separately, heterozygotes were still significantly increased in DR3+ Graves' disease patients (54 of 77, 70%) compared to DR3+ controls (21 of 45, 47%; chi 2 = 6.6, P less than 0.04). Furthermore, TNF fragment heterozygotes were found predominantly in patients, who had TSH-receptor antibodies (29/45, 64%, P less than 0.007), implying that these patients might represent an immunogenetic subset of the disease. Although TNF beta polymorphisms are linked to A1B8DR3, these results suggest that they represent an additional susceptibility marker in Graves' disease.

Effects of pioglitazone in nondiabetic patients with arterial hypertension: a double-blind, placebo-controlled study.

Hypertension is often associated with insulin resistance, dyslipidemia and obesity, which indicate a prediabetic state and increased risk of cardiovascular disease. Pioglitazone treatment of patients with type 2 diabetes reduces insulin resistance and improves lipid profiles. The present double-blind placebo-controlled study is the first study to report effects of pioglitazone in non-diabetic patients with arterial hypertension. Following a one week run-in, 60 patients were randomized to receive either pioglitazone (45 mg/day) or placebo for 16 weeks. Insulin sensitivity (M-value) increased by 1.2 +/- 1.7 mg/min/kg with pioglitazone compared with 0.4 +/- 1.4 mg/min/kg (P = 0.022) with placebo. HOMA index was decreased (-22.5 +/- 45.8) by pioglitazone but not by placebo (+0.8 +/- 26.5; P < 0.001). Decreases in fasting insulin and glucose were significantly (P = 0.002 and P = 0.004, respectively) greater with pioglitazone than placebo. Body weight did not change significantly with either treatment. HDL-cholesterol was increased and apolipoprotein B was decreased to a significantly greater extent with pioglitazone. There was a significantly (P = 0.016) greater decrease from baseline in diastolic blood pressure with pioglitazone. These changes would suggest improved glucose metabolism and a possible reduction in risk of cardiovascular disease with pioglitazone treatment of non-diabetic patients with arterial hypertension.

Codon 17 polymorphism of the cytotoxic T lymphocyte antigen 4 gene in Hashimoto's thyroiditis and Addison's disease.

Endocrine autoimmune disorders share susceptibility and resistance factors of the human leukocyte antigen system on the short arm of chromosome 6, but other gene loci also contribute to predisposition and protection. Because the cytotoxic T lymphocyte antigen 4 (CTLA4) alanine-17 encoded by the CTLA4 gene on chromosome 2q33 confers susceptibility to Graves' disease, as well as to type 1 (insulin-dependent) diabetes mellitus, we investigated this dimorphism in the other endocrine autoimmune disorders: Hashimoto's thyroiditis and Addison's disease. We analyzed the CTLA4 exon 1 polymorphism (49 A/G) in 73 patients with Hashimoto's thyroiditis, 76 with Addison's disease, and 466 healthy controls. This dimorphism corresponds to an aminoacid exchange (Thr/Ala) in the leader peptide of the expressed protein. CTLA4 alleles were defined by PCR, single-strand conformational polymorphism analysis, and restriction fragment length polymorphism analysis using BbvI. Patients with Hashimoto's thyroiditis had significantly more Ala alleles than controls, both as homozygotes (22% vs. 15%) and heterozygotes (53% vs. 46%), and less Thr than controls as homozygotes (25% vs. 39%), P < 0.04. The phenotypic frequency for Ala was significantly higher in patients (75%), compared with controls (61%), P < 0.03. Patients with Addison's disease did not differ significantly from controls, but those carrying the suceptibility marker, human leukocyte antigen DQA1*0501, were significantly more CTLA4 Ala17 positive than controls with the same DQA1 allele (P < 0.05). In conclusion, an alanine at codon 17 of CTLA4 confers genetic susceptibility to Hashimoto's thyroiditis, whereas this applies only to the subgroup of DQA1*0501+ patients with Addison's disease.

CTLA4 alanine-17 confers genetic susceptibility to Graves' disease and to type 1 diabetes mellitus.

The genetic susceptibility to Graves' disease and type 1 (insulin-dependent) diabetes mellitus is conferred by genes in the human leukocyte antigen region on the short arm of chromosome 6, but several other genes are presumed to determine disease susceptibility. Among those candidate genes is the cytotoxic T lymphocyte antigen 4 (CTLA4) located on chromosome 2q33 in man. We investigated the distribution of the CTLA4 exon 1 polymorphism (49 A/G) in Graves' disease and IDDM. This dimorphism at codon 17 results in an amino acid exchange (Thr/Ala) in the leader peptide of the expressed protein and was analyzed by PCR, single strand conformation polymorphism, and restriction fragment length polymorphism analysis in 305 patients with Graves' disease, 293 patients with IDDM, and 325 controls. Patients with Graves' disease had significantly more Ala alleles than controls, both as homozygotes (21% vs. 13%) and as heterozygotes (53% vs. 46%), and less Thr as homozygotes (26% vs. 42%; P < 2 x 10(-4). The phenotypic frequency of Ala-positive patients (73%) was significantly higher than of controls (58%; P = 10(-4); relative risk = 2). Patients with IDDM also had significantly more Ala alleles as homozygotes (19%) or heterozygotes (50%; P = 0.01). In conclusion, an alanine at codon 17 of CTLA4 is associated with genetic susceptibility to Graves' disease as well as to IDDM.