Enhancement of glucose transport by selected plant foods in muscle cell line L6.

Glucose uptake activity of 11 plant foods was assessed in L6 myotubes. Among them onion and ginger showed potent enhancement of glucose transport. This effect required new protein synthesis of glucose transporters. In addition, onion-induced glucose uptake in L6 myotubes was mediated through activation of phosphoinositide 3-kinase.

Analysis of the thyrotropin receptor as a candidate gene in familial Graves' disease.

Familial clustering of Graves' disease indicates a genetic etiology. Searches for genetic factors additional to the known human leukocyte antigen (HLA) association have implicated the gene for the TSH receptor (TSHR). We analyzed the linkage and association among three recently described microsatellite markers within the TSHR introns in Graves' disease in large multiply affected Welsh and English families (223 members, 44 affected individuals). Linkage analysis under a dominant model strongly rejected the hypothesis that TSHR is linked to Graves' disease in these families (lod score = -4.53). More detailed analyses also failed to provide evidence for linkage; these included combined segregation and linkage analysis, correction for HLA-DR3 status, allowance for the levels of thyroid autoantibodies in unaffected pedigree members, consideration of a recessive model for the disease, and linkage disequilibrium between disease and marker alleles. We also considered the possibility of a genetic heterogeneity of Graves' disease and thus analyzed separately the different families with a similar result. Although these results cannot eliminate a minor role of the TSHR gene locus in the genetics of Graves' disease, they argue against it being a major genetic determinant in this pathology.

Combined segregation and linkage analysis of Graves disease with a thyroid autoantibody diathesis.

Combined segregation and linkage analysis is a powerful technique for modeling linkage to diseases whose etiology is more complex than the effect of a well-described single genetic locus and for investigating the influence of single genes on various aspects of the disease phenotype. Graves disease is familial and is associated with human leukocyte antigen (HLA) allele DR3. Probands with Graves disease, as well as close relatives, have raised levels of thyroid autoantibodies. This phenotypic information additional to affection status may be considered by the computer program COMDS for combined segregation and linkage analysis, when normals are classified into diathesis classes of increasing thyroid autoantibody titer. The ordinal model considers the cumulative odds of lying in successive classes, and a single additional parameter is introduced for each gene modeled. Distributional assumptions are avoided by providing estimates of the population frequencies of each class. Evidence for linkage was increased by considering the thyroid autoantibody diathesis and by testing two-locus models. The analysis revealed evidence for linkage to HLA-DR when the strong coupling of the linked locus to allele DR3 was considered (lod score of 6.6). Linkage analysis of the residual variation revealed no evidence of linkage to Gm, but a suggestion of linkage to Km.

HLA-DPB1 polymorphisms on the MHC-extended haplotypes of families of patients with Graves' disease: two distinct HLA-DR17 haplotypes.

HLA-DPB1 polymorphisms were investigated in 217 members of 21 multiplex families of patients with Graves' disease, who had previously been haplotyped, using in vitro enzymatic DNA amplification and hybridization with sequence-specific oligonucleotide probes. Using the strategy of group-specific amplification, we were able to assign 19 DPB1 alleles with the use of 13 sequence specific oligonucleotide probes. No recombination was found between HLA-DPB1 and the HLA-DR/DQ complex in 243 informative meioses. HLA-DPB1*0401 was found to be the most common allele followed by DPB1*0101, *0201 and 0402 with allele frequencies of 0.4214, 0.1132, 0.1069 and 0.1006, respectively. Besides the strong linkage disequilibrium between HLA-DPB1*0101 allele and the HLA-B8, DR17/DQ2 haplotype; HLA-DPB1*0202 and *1101 alleles were also found to be significantly associated with HLA-B18 and DR7 with Pc < 0.001 and Pc < 0.009, respectively. HLA-DR17/DQ2 was found to be more commonly associated with HLA-DPB1*0101 on the Affected haplotypes (from family members affected with Graves' disease) while associated with DPB1*0401 on the Ab + ve haplotypes (deduced from thyroid autoantibody positive unaffected members). The differences in the frequency of this preferential association on the Affected and Ab + ve haplotypes was statistically significant (ch 2 = 10.18, df = 2, P < 0.007). Though it is rater unlikely that the HLA-DPBI polymorphism by itself could contribute a direct role towards the genetic susceptibility for the development of autoimmune thyroid disease, it could serve as a marker in identifying family members with the HLA-DR17/DQ2 haplotype who are more likely to develop Grave's disease or thyroid autoantibodies.

HLA-DPB1 polymorphisms in patients with hyperthyroid Graves' disease and early onset myasthenia gravis.

Using the technique of in vitro enzymatic DNA amplification and dot blot hybridization with sequence-specific oligonucleotide (SSO) probes, a study of genetic polymorphism of HLA-DPB1 was performed in 83 unrelated patients with Graves' disease (GD), 48 patients with early onset myasthenia gravis (EOMG) and 100 normal British caucasoid subjects who were also tissue typed for HLA-A, B and DR antigens. HLA-DPB1*0401 was the commonest allele in both patient and control groups with gene frequencies of 0.380, 0.333 and 0.445 for GD, EOMG and controls, respectively. No significant independent association was found with any HLA-DPB1 allele. As expected, HLA-DR17 is significantly associated with Graves' disease (pc < 8 x 10(-3), RR = 2.9), while both HLA-B8 and DR17 are significantly associated with EOMG (pc < 2 x 10(-7), RR = 10.3 and pc < 0.02, RR = 3.4, respectively)] HLA-DR2 is also significantly increased in EOMG patients who were negative for HLA-DR17 (pc < 0.02, RR = 6.4). In addition, the co-occurrence of HLA-B8 with DPB1*0402 was significantly commoner in patients with GD (p < 0.021, RR = 6.2) and EOMG (p < 0.0007, RR = 10.8) than in controls, although the HLA-DPB1*0402 by itself showed no significant increase.

MHC-extended haplotypes in families of patients with Graves' disease.

MHC-extended haplotypes were investigated in multiplex families of patients with hyperthyroid GD. Using a combination of both phenotypic (serology and protein electrophoresis) and genotypic (DNA-RFLP) markers, 159 MHC-extended haplotypes extending from HLA-A across the MHC class III (C2, Bf, C4A, and C4B) toward the HLA-DR/DQ complex were deduced from 217 (51 and 166 affected and unaffected) members of 21 families of patients with GD. Thyroid autoantibodies were measured and found positive in 27.1% of 166 clinically euthyroid unaffected members. Extended haplotypes were classified into four categories--affected (n = 40), Aff/Ab + ve (shared haplotype between affected and Ab + ve members, n = 31), Ab + ve (n = 29), and Ab - ve (n = 59)--based on the presence and absence of these haplotypes in 51 affected members with GD and 45 and 121 unaffected members who were respectively positive and negative for thyroid autoantibodies. Five recombinations were detected: three were found between HLA-A and B and two between HLA-B and the MHC class III. No recombination was found between or within the MHC class III and class II complex. Though the HLA-DR17 (DR beta 17(1) and DR beta 17(2)) allele was found to be significantly increased in both the affected and the Aff/Ab + ve when compared with the Ab - ve haplotypes (p < 0.042 and p < 0.018), the frequency of the HLA-B8, 2.7-kb SstI-4.5-kb TaqI/C2 Bf*S, 6.4-kb TaqI/C4A*Q0C4B*1, HLA-DR beta 17(1)/DQ alpha 2-DQ beta 2a extended haplotype was found to be significantly increased only in the affected haplotype (p < 0.05). These results suggest that while HLA-DR17 is a susceptibility allele shared between GD and individuals with positive thyroid autoantibodies, the HLA-B8, 2.7-kb SstI-4.5-kb TaqI/5'-3'C2 Bf*S, 6.4-kb TaqI/C4A*Q0B*1, DR beta 17(1)/DQ alpha 2-DQ beta 2a is a disease susceptibility-extended haplotype for Graves' disease.

Antigen-specific T cell recognition of affinity-purified and recombinant thyroid peroxidase in autoimmune thyroid disease.

The T cell proliferative responses of peripheral blood lymphocytes from 20 patients with autoimmune thyroid disease (AITD) and 20 healthy controls were analysed to immunoaffinity-purified thyroid peroxidase (TPO) and recombinant antigen preparations generated in Escherichia coli as glutathione-s-transferase fusion proteins. The epitope specificity of the T cell response was investigated using a selection of eight discrete recombinant fragments encompassing the whole of the extracellular region of the TPO molecule. Significant differences in the proliferative responses between patients and controls were observed to the full length, affinity-purified TPO molecule (P less than 0.002) as well as to the recombinant fragments R1c (residues 145-250) (P less than 0.001) and R2b (residues 457-589) (P less than 0.001) suggesting the presence of at least two distinct T cell determinants on this autoantigen. One of these T cell epitopes, localized within the region R1c, has not previously been identified by studies with synthetic peptides.

Heat shock protein 70 (HSP70) and complement C4 genotypes in patients with hyperthyroid Graves' disease.

The genetic polymorphisms of the heat shock protein 70 (HSP70) and complement component C4 were investigated in 90 patients with hyperthyroid Graves' disease and 92 normal control subjects. The 8.5-kb PstI HSP70 allele was strongly associated with Graves' disease when compared with controls (P less than 0.001). The presence of the 8.5-kb PstI HSP70 allele was strongly associated with a deletion of the C4A gene in both patients and controls (P less than 0.0003 and P less than 0.00005 respectively). However, in the absence of C4A gene deletion, the frequency of the 8.5-kb PstI HSP70 allele was still significantly higher in patients when compared with controls (P less than 0.04). These results suggest that the HSP70 locus may have an immunological role to play in autoimmune hyperthyroid Graves' disease.

Linkage disequilibrium between the HLA-DQA2 alleles and the HLA/DR/DQ complex.

A statistically significant association was observed between alleles of the HLA-DQA2 and of the DR/DQ complex in a DNA-restriction fragment length polymorphism study of 219 members from 21 multiplex families of patients with hyperthyroid Graves' disease and 773 unrelated individuals selected for homozygosity of the HLA-DQA2 alleles. This data provides evidence for linkage disequilibrium rather than for a hot spot of recombination within the HLA-DQ subregion.

DQA2 U allele: a genetic marker for relapse of Graves' disease.

The association of HLA-DR3 with Graves' disease in Caucasoids is well established but its significance is unclear and its clinical value as a predictive parameter for relapse after a course of antithyroid drug therapy is controversial. We have further investigated the predictive value at the genomic level in 51 patients with Graves' disease who were treated with a 6-month course of carbimazole and followed up for 2 years. Using DNA-restriction fragment length polymorphism (DNA-RFLP) allogenotyping, (i) complete concordance of HLA-DR assignment was observed between serological and DNA-RFLP analysis of all but one of 51 patients with Graves' disease; (ii) the DR beta 17(1)-DQ alpha 2-DQ beta 2a (a DNA-RFLP allogenotype of the classical Northern European haplotype of HLA-B8 DR3) was significantly (corrected P = Pcorr less than 0.02) associated with Graves' disease particularly in patients who relapsed (Pcorr less than 0.005); (iii) HLA-DR3 was highly associated with DQA2 U allele (chi 2 = 18.53, d.f.2, P less than 0.0005); (iv) a strong correlation between the DQA2 U allele and the outcome of the disease was observed. Relapse occurred in 91% (10/11) of the patients who were homozygous for the DQA2 U allele whilst only 65% (15/23) and 41% (7/17) of patients who were hetero or homo-zygous for the DQA2 L allele (DQA2 U/L and DQA2 L/L) relapsed within the same period of follow-up (chi 2 = 7.18, d.f.2, P less than 0.05). Though the relapse rate in patients with the DQA2 U/U genotype was not significantly higher than the relapse rate in patients with the DQA2 U/L genotype, it was significantly higher than the relapse rate in patients with DQA2 L/L genotype (P less than 0.0001) with a relative risk of 14.3.

Complement C4 and heat shock protein 70 (HSP70) genotypes and type I diabetes mellitus.

Type I diabetes is strongly associated with the major histocompatibility complex (MHC) class II region (DR and DQ loci), and to a lesser extent the class III region (complement C4 loci). Restriction fragment length polymorphism analysis was employed to investigate the C4 and heat shock protein 70 (HSP70) loci of 176 patients with type I diabetes and 92 healthy controls. In the patient population there was an excess of deletions of the C4A locus (48.5% vs 22.1%, P less than 0.0005). The HSP70 probe in conjunction with the restriction endonuclease Pst I detects two alleles of 9 or 8.5 kilobases (kb). The 8.5 kb allele was significantly increased in the patient group compared to healthy controls (0.569 vs 0.353, respectively, P less than 0.0005). Furthermore, a C4A deletion nearly always occurred with the 8.5 kb HSP70 allele, suggesting that it may be a marker of the HLA-A1,B8,C4A deletion, DR3 extended haplotype.

Enhanced resolution of the SstI polymorphic variants of the C2 locus: description of a new size class.

Investigation of the SstI polymorphism at the 5' region of the gene encoding the serum complement component, C2, in unrelated individuals has been limited by inadequate resolution of the different size variants using conventional agarose gel electrophoresis. We have overcome this problem by the simple modification of electrophoretic conditions using both low current and temperature. With the technique described in this report it has been possible to assign the hetero- and homozygosities of the SstI polymorphism in unrelated individuals without the need for family studies. Using this approach we have investigated a population of 60 healthy, unrelated Caucasians. Five different variants of the SstI polymorphism, the 2.75-, 2.7-, 2.65-, 2.55-, and 2.4-kb fragments, were observed. The 2.7-kb SstI fragment is the most common variant, followed by the 2.65- and the less common 2.4-kb SstI fragments. These fragments occurred at frequencies of 53.3, 35.8, and 7.5%, respectively. We report here a new rare variant of the 2.75- and another rare variant of the 2.55-kb SstI fragments; both occurred at a frequency of 1.7%. The latter variant was previously described as the 2.6-kb SstI fragment but is reclassified in this report as 2.55-kb, based on the results of better resolution on electrophoresis. All the larger-size classes of the SstI polymorphic variants, the 2.75-, 2.7-, and 2.65-kb fragments, are associated with the 4.5-kb TaqI fragment, while the smaller-size classes of 2.55- and 2.4-kb fragments are associated with the 6.6-kb TaqI fragment at the 3' end of the C2 gene.

C4A gene deletion: association with Graves' disease.

The association of HLA class I and class II antigens, particularly HLA-B8,DR3, with a variety of autoimmune diseases has been well documented. The C4A*Q0 (non-expressed C4A) allele which is in linkage disequilibrium with HLA-B8,DR3 has also been reported to be associated with systemic lupus erythematosus, insulin-dependent diabetes mellitus and Graves' disease. However, the number of studies has been limited by the requirement of family data for the assignment of the C4A*Q0 allele based on C4 protein typing. Recently, with the availability of a C4 cDNA probe, a C4A gene deletion associated with HLA-B8,DR3 has been reported in normal individuals. We have tried to resolve the problem of assigning the C4A*Q0 allele by using both phenotypic and genotypic approaches and have determined the significance of the C4A*Q0 allele in 80 unrelated patients with Graves' disease and in 50 normal control subjects. Our results demonstrate a strong association of the C4A*Q0 allele with Graves' disease (56 versus 26%; P less than 0.002, relative risk = 3.7) and in particular in association with HLA-B8 and/or DR3 (92 versus 70.6%; P less than 0.04) when compared with normal controls. All the C4A*Q0 alleles that were associated with HLA-B8 and/or DR3 were due to a C4A gene deletion. Of the C4A*Q0 alleles, in Graves' disease, 94% (compared with 82% in the control group) could be detected by C4 DNA analysis using either TaqI or EcoRI restriction endonucleases. It is suggested that a combination of C4 protein typing with C4 DNA analysis is the best approach for the determination of the C4A*Q0 allele in unrelated individuals without access to family data.

Thyroglobulin antibodies in Graves' disease are associated with T-cell receptor beta chain and major histocompatibility complex loci.

We have investigated the T-cell antigen receptor constant beta and alpha chain genes (TCR-C beta, -C alpha) and the immunoglobulin (Ig) heavy chain switch regions of patients with Graves' disease (GD) using restriction fragment length polymorphism (RFLP) analysis. No significant associations were found with RFLPs of either the TCR-C beta, -C alpha or Ig heavy chain switch region loci and GD. However, a significant association was found between the presence of anti-thyroglobulin (anti-Tg) antibodies in the serum of patients and the 10.0; 9.2 kb TCR-C beta genotype (P less than 0.02). Also, those patients with anti-Tg antibodies had an increased frequency of HLA-DR3 (P less than 0.025). These results suggest that genes residing in the TCR chain and major histocompatibility complex loci may be important in determining the immune response to thyroglobulin but not to the disease itself.

Complement component C9 in Graves' disease.

C9, the terminal component of complement, is the key part of the membrane attack complex formed as a result of complement activation; it has also been reported to be an acute phase protein. Its potential role in Graves' disease has been studied by measuring plasma C9 concentrations using an automated two-site immunoradiometric assay employing monoclonal antibodies, whose binding to thyroid tissue has also been investigated. The plasma C9 concentration in patients with hyperthyroid Graves' disease (86.3 +/- 21.6 mg/l, mean +/- SD; n = 49) was significantly increased (P less than 0.001) compared with normal subjects (60.4 +/- 13.4 mg/l; n = 48). In contrast, the plasma concentration of C-reactive protein, a marker of the acute phase response, was not significantly different between the two groups. The plasma C9 concentration in patients with hyperthyroid Graves' disease decreased significantly (P less than 0.01) after treatment with antithyroid drugs (carbimazole or methimazole; n = 14), but not after radioactive iodine (131I) treatment (n = 18). Immunohistochemical staining demonstrated that monoclonal antibody to C9 bound to the basement membranes of thyroid follicular cells of Graves' thyroid tissue but not to normal thyroid tissue. Radiolabelled monoclonal antibody to C9 bound to membrane fragments prepared from thyroid glands from two patients with Graves' disease. We conclude that C9, and by implication the membrane attack complex, may be involved in the pathogenesis of Graves' disease.

Prediction of outcome in Graves' disease after carbimazole treatment.

In a prospective study to determine which factors would predict remission or relapse, 65 patients with hyperthyroid Graves' disease were treated for six months with a blocking replacement regimen of carbimazole, 40 mg daily, and triiodothyronine (T3). They were followed for one year after stopping treatment, by which time 32 (49 per cent) had relapsed. Although the treatment protocol, relapse rate and frequency of the HLA-DR3 antigen in this population were similar to those of a regionally separate Graves' population investigated previously, the predictive value of HLA-DR3 status together with thyroid stimulating antibody (TSAB) levels was strikingly different. In the present study there was no significantly abnormal distribution of any HLA antigen in the relapse group compared with those patients who achieved remission. Thyroid stimulating antibodies were detected in 62 patients (95 per cent) and fell significantly (p less than 0.05) after carbimazole treatment, irrespective of DR3 status or outcome; TSAB levels only became undetectable in nine patients (28 per cent) who subsequently relapsed and in nine patients (30 per cent) who maintained remission. T3-suppressed 20 min 123I uptake fell equally after treatment in the relapse and remission groups but continued to fall thereafter in the group which maintained remission. In these patients, 123I uptake was significantly lower at the end of the study period than at the end of treatment (p less than 0.05). Serum free T4 levels were higher before treatment in the patients who later relapsed than in those whose disease remitted (p less than 0.02). This proved the only significant marker associated with outcome but was of little predictive value in any patient. This study highlights the problem in predicting the outcome of antithyroid drug treatment, since even within the same country under similar conditions, divergent results have been obtained. It appears that the loci controlling the immune response in Graves' disease are likely to include genes lying outside the HLA-DR region. The results also suggest that the immunological effects of antithyroid drugs are maintained after stopping treatment in those patients whose disease remits.

Reticuloendothelial Fc function in normal individuals and its relationship to the HLA antigen DR3.

Reticuloendothelial Fc function was examined in 24 normal individuals by determining the clearance rates of erythrocytes coated with rhesus antibody (IgG RBC) or chemically modified by treatment with n-ethyl malemide (NEM). The mean clearance times for the IgG RBC (52.1 +/- 10.3 min) and the NEM cells (87.5 +/- 14.7 min) in the 10 individuals who possessed the HLA antigen DR3 did not differ significantly from the mean clearance times observed in the 14 non-DR3 controls (IgG RBC 51.5 +/- 6.9 min and NEM cells 77.6 +/- 4.5 min). Two individuals with prolonged clearance times for the IgG RBC were identified in each group, the values observed being 80, 103, 119 and 137 min. These prolonged clearance times could not be attributed to lower numbers of antibody molecules bound to the red cell surface. The prolonged clearance times were reproducible but the putative Fc receptor defect in these individuals could be abolished by using IgG RBC obtained from different donors. When IgG RBC, prepared from a single donor were used to assess Fc function, no difference in the clearance rates of DR3 positive and DR3 negative individuals was found. Our results indicate that differences in the distribution of the rhesus antigen on the red cell surface can markedly influence the fate of these cells in vivo. We suggest that this is the explanation for our failure to confirm the association between Fc receptor defects and the HLA antigen DR3.

Failure to demonstrate cell-mediated immune responses to thyroid antigens in Graves' disease using in vitro assays of lymphokine-mediated migration inhibition.

The use of in vitro assays of cell-mediated immunity has provided evidence of a defect in thyroid antigen-specific T suppressor cells in the peripheral blood of patients with autoimmune diseases. We employed a direct assay of T lymphocyte migration inhibition in an attempt to demonstrate cell-mediated immune responses to thyroid antigens in untreated patients with hyperthyroid Graves' disease and examined the influence of (1) a variety of antigen preparations, (2) different assay conditions, and (3) the HLA-DR3 status of normal subjects on this system. There was no significant difference in the migration of lymphocytes from 13 untreated patients with hyperthyroid Graves' disease to a crude 800 X g supernatant antigen preparation of normal human thyroid when incubated at either 20 or 37 C compared with the response of 13 normal subjects. The mean migration index was 57 +/- 22 (+/- SD) in the Graves' patients compared with 65.2 +/- 19.9 in the normal subjects at 20 C and 83 +/- 16.9 in the Graves' patients compared with 86.6 +/- 15.1 in the normal subjects at 37 C. Similarly, there was no significant difference in the migration indices obtained with T cells from Graves' patients and normal subjects using an 800 X g supernatant prepared from the thyroid glands of 2 patients with Graves' disease, incubated at 20 or 37 C (44.0 +/- 22.5 in the Graves' patients compared with 45.7 +/- 12.8 in the normal subjects at 20 C and 67.9 +/- 20.8 in the patients compared with 72 +/- 12.6 in the controls at 37 C). In contrast, the migration indices calculated for 20 C incubation were significantly lower than the corresponding value at 37 C using 800 X g normal thyroid antigen (P less than 0.05) and 800 X g Graves' thyroid antigen (P less than 0.01) in both patient and control groups. An identically prepared uterine antigen produced a similar reduction in the migration indices at 20 C compared with those at 37 C in 6 normal donors (P less than 0.05), but no temperature effect was found when 8 normal subjects were tested with purified protein derivative of tuberculin. The responses of 12 normal individuals to 800 X g supernatants of the normal and Graves' thyroid antigens were not influenced by the HLA-DR3 antigen.(ABSTRACT TRUNCATED AT 400 WORDS)