Hajime Tamai

Association of the T-cell regulatory gene CTLA4 with Graves’ disease and autoimmune thyroid disease in the Japanese

AbstractAutoimmune thyroid disease (AITD) is caused by an immune response to self-thyroid antigen. The cytotoxic T-lymphocyte antigen-4 (CTLA4) gene, encoding a negative regulator of the T-lymphocyte immune response, had been reported to be associated and/or linked to AITD. Recently, AITD susceptibility in the Caucasians was mapped to the 6.1-kb 3′UTR of the CTLA4 gene, in which the three single-nucleotide polymorphisms (SNPs) CT60, JO31, and JO30 were strongly associated with AITD. In order to determine the association of the CTLA4 gene with AITD in the Japanese, case-control association analysis for the four SNPs of the CTLA4 gene using 380 AITD patients and 266 healthy controls was done. Among the SNPs examined, the SNP JO31 was most significantly associated with AITD in the Japanese, whereas the association of the JO30 with AITD was not observed. The frequency of the disease-susceptible G allele of the JO31 of the Japanese control was higher than that of the Caucasians (67.1% vs 50.2%); however, the G allele of the JO31 was associated with Graves′ disease (GD) (67.1% vs 76.3%, P=0.0013) and AITD in the Japanese (67.1% vs 74.2%, P=0.0055). Furthermore, the G allele of the JO31 was associated with the increased risk for GD [ P=0.0051, odds ratio (OR)=1.7] and AITD (P=0.016, OR=1.5) in a dominant model. These results suggested that the CTLA4 gene is involved in the susceptibility for GD and AITD in the Japanese.

Association between the DRB1*08032 Histocompatibility Antigen and Methimazole-Induced Agranulocytosis in Japanese Patients with Graves Disease

Objective: To determine the association between HLA class II genes and methimazole-induced agranulocytosis in patients with Graves disease. Design: Case-control study. Setting: Kuma Hospital, which specializes in thyroid diseases, in Kobe, Japan. Subjects: 24 patients with Graves disease who had methimazole-induced agranulocytosis diagnosed by peripheral granulocyte counts of less than 0.5 109/L, and 68 patients with Graves disease treated with methimazole, who were free from agranulocytosis. Controls were 525 healthy, unrelated Japanese student volunteers at Kyushu University in Japan. Measurements: All HLA class II genes were analyzed for polymorphisms at the DNA level by using the polymerase chain reaction sequence-specific oligonucleotide probes method. The allele frequencies in the agranulocytotic Graves disease group were compared with those in the nonagranulocytotic Graves disease and control groups. Results: A strong positive association was seen in DRB1*08032 between the agranulocytotic group and both the control and nonagranulocytotic Graves disease groups. Conclusion: The HLA DRB1*08032 allele was strongly associated with susceptibility to methimazole-induced agranulocytosis, suggesting that cellular autoimmunity may be involved in its development. The thioureylene antithyroid drugs methimazole and propylthiouracil have been widely used to treat Graves disease [1], but side effects are found in 1% to 5% of treated patients [2-4]. One important and serious side effect of this treatment is agranulocytosis, which occurs in 0.1% to 0.3% of treated patients [5-10]. Although the mechanisms responsible for the agranulocytosis are unclear, an immune phenomenon may be involved, because antigranulocyte antibodies [11-20] or lymphocyte sensitization to antithyroid drugs [17] are found in agranulocytotic patients. These autoantibodies may induce agranulocytosis by direct cytotoxicity or through blocking of membrane proteins that are important for the maturation of progenitor cells [19]. Antibodies are generally produced by plasma cells, which are maturated from B lymphocytes in the presence of various cytokines produced by activated CD4+ T lymphocytes and by direct T lymphocyte-B lymphocyte interactions [21]. CD4+ T lymphocytes are activated when they encounter antigenic peptide with major histocompatibility complex class II on antigen-presenting cells, such as B lymphocytes and macrophages. The recognition of major histocompatibility complex class II peptide complexes by T lymphocytes is therefore central to the development of immune responses and antibody production. The major histocompatibility complex class II molecules are highly polymorphic heterodimeric membrane glycoproteins composed of and chains. In humans, at least three major histocompatibility complex class II moleculesHLA-DR, HLA-DQ, and HLA-DP antigensare expressed on the surface of antigen-presenting cells. In addition, these molecules are encoded by the genes in the HLA region: DRA, DQA, and DPA for chains and DRB, DQB, and DPB for chains of each HLA molecule. The function of major histocompatibility complex class II molecules is to bind short peptides derived mainly from extracellular proteins, in turn forming major histocompatibility complex class II peptide complexes that interact with appropriate T-cell receptors of CD4+ T lymphocytes [22]. Each major histocompatibility complex class II molecule binds different sets of proteolytic fragments of peptides, thus contributing to the diversity of immune responsiveness among individual persons [23-26]. Identification of susceptible HLA class II antigens to antithyroid drug-induced agranulocytosis is essential to understanding the development of the disorder [27, 28]. Methods Participants We studied 24 patients with Graves disease and methimazole-induced agranulocytosis, 68 patients with Graves disease and no agranulocytosis who were treated with methimazole, and 525 healthy unrelated student volunteers from Kyushu University. The agranulocytotic group consisted of 5 males and 19 females ranging in age from 16 to 68 years (mean, 38.4 years). Agranulocytosis was defined as peripheral granulocyte counts less than 0.5 109/L. The nonagranulocytotic Graves disease group consisted of 10 males and 58 females with ages ranging from 11 to 58 years (mean, 32.4 years). Graves disease was diagnosed on the basis of clinical symptoms, thyroid function test results, positivity for thyroid-stimulating hormone-binding inhibitory immunoglobulin, and 123I uptake. Blood samples were taken after informed consent was obtained. DNA Typing of HLA Class II Genes We extracted DNA from peripheral granulocytes using a previously described method [28]. Genomic DNA was subjected to 30 cycles of polymerase chain reaction in a thermal cycler (Perkin Elmer Cetus, Norwalk, Connecticut) to amplify the second exons of the DRB1, DQA1, DQB1, DPA1, and DPB1 genes using thermostable DNA polymerase (Ampli Taq, Perkin Elmer Cetus) [29]. The primers and sequence-specific oligonucleotide probes for the HLA-DNA typing were previously reported [29]. We used the nomenclature of HLA alleles recommended by the official nomenclature committee [30, 31]. Statistical Analysis We compared frequencies of HLA-DRB1, DQA1, DQB1, DPA1, and DPB1 alleles in the agranulocytotic group with those in the control and nonagranulocytotic Graves disease groups. Strength of the statistical association between the disease and HLA allele was expressed by the odds ratio, and the statistical significance was examined by using the chi-square test with Yates correction. We calculated the corrected P value by multiplying the P value by the number of alleles tested for each class II gene (58 DRB1 alleles, 13 DQA1 alleles, 14 DQB1 alleles, 6 DPA1 alleles, and 46 DPB1 alleles). Results HLA Class II Alleles Associated with the Agranulocytotic Group We observed positive associations between HLA class II alleles and agranulocytosis in DRB1*1501 (37.5% phenotype frequencies in the patients with agranulocytosis compared with 12.3% in controls; odds ratio, 4.25; P = 0.001), DRB1*08032 (54.2% compared with 17.9%; odds ratio, 5.42; P < 0.001), DQA1*0103 (66.7% compared with 40.4%; odds ratio, 2.95; P = 0.02), DQB1*0602 (33.3% compared with 11.8%; odds ratio, 3.73; P = 0.006), and DPB1*0501 (95.8% compared with 60.6%; odds ratio, 15.0; P = 0.001) (Table 1). The positive associations between HLA class II alleles and agranulocytosis in both DRB1*08032 and DPB1*0501 remained statistically significant, even when the P value was corrected by multiplying the number of tested alleles in each locus. We believe that the relatively increased frequencies of DQB1*0602 and DQA1*0103 were caused by linkage disequilibria between DRB1 and DQ alleles, because DRB1*1501-DRB5*0101-DQA1*0102-DQB1*0602 and DRB1*0803-DQA1*0103-DQB1*0601 haplotypes are frequently found in Japanese persons (11.6% and 17.9%, respectively) [32]. Table 1. Frequencies of HLA Class II Alleles in Patients with Agranulocytosis and in Controls Identification of HLA Alleles Specific for Susceptibility to Agranulocytosis Underlying Graves disease is a typical organ-specific autoimmune disease, and the association between the disease and HLAs has been reported in various ethnic groups [33-34]. We have reported that the susceptibility to Graves disease in Japanese persons is strongly associated with DPB1*0501 [35] and that resistance to the disease is associated with DQB1*0501 [36]. To distinguish genes associated with susceptibility to agranulocytosis from those associated with Graves disease, we compared frequencies of the HLA alleles associated with agranulocytosis in patients with Graves disease with those in nonagranulocytotic patients with Graves disease. Table 2 lists allele frequencies in the agranulocytotic and nonagranulocytotic Graves disease groups. The frequency of the DRB1*08032 allele was significantly increased in the agranulocytotic group (frequency of 54.2% in the agranulocytotic group compared with 22.1% in the nonagranulocytotic group; odds ratio, 4.18; P = 0.007). We found no significant difference in the frequencies of DRB1*1501 and DPB1*0501 between the two groups, although both alleles were more frequent in the agranulocytotic group than in the nonagranulocytotic group. Table 2. Allele Frequencies in Agranulocytotic and Nonagranulocytotic Patients with Graves Disease Discussion The HLA class II oligotyping data that we report suggest that the susceptibility to methimazole-induced agranulocytosis in patients with Graves disease has a genetic factor. In Japanese persons, methimazole-induced agranulocytosis shows strong associations with the presence of DRB1*08032 and DPB1*0501. Among these alleles, the frequency of DRB1*08032 was increased significantly in agranulocytotic patients compared with patients without agranulocytosis, suggesting that DRB1*08032 specifically is associated with susceptibility to agranulocytosis. The DR8 (DR -DRB1*08032) antigen is probably directly involved in the development of methimazole-induced agranulocytosis in Japanese persons, although other genes in close linkage disequilibrium with HLA-DRB1*08032 may also be involved [37]. We previously reported an association between the susceptibility to Graves disease and DPB1*0501 in Japanese persons [35], and the strong association with agranulocytosis (in comparison with controls) observed in this study may reflect the association between the allele and susceptibility to Graves disease. However, we could not exclude the possibility that DPB1*0501 was also involved in susceptibility to agranulocytosis, because the odds ratio for this allele was 3.51 compared with nonagranulocytotic patients (Table 2). The misconception that the increased frequency of DPB1*0501 was not significant may have originated because this allele is common in patients with Graves disease (the frequency of DPB1*0501 in nonagranulocytotic patients with Graves disease was

Independent Involvement of CD8+CD25+ Cells and Thyroid Autoantibodies in Disease Severity of Hashimoto's Disease

Hashimotos disease (HD) is well known as an autoimmune thyroid disease caused by the destruction of the thyroid follicles, and can be diagnosed in the subclinical stage with thyroid-specific autoantibodies. However, some patients with HD develop hypothyroidism and are treated with thyroxine (severe HD), but most do not throughout their lives (mild HD). To clarify the immunologic differences between these two groups of patients with HD, we examined serum thyroid autoantibodies (antithyroid peroxidase antibodies and antithyroglobulin antibodies), CD4+ CD25+ cells that contain regulatory T cells and activated helper T cells, and CD8+ CD25+ cells that are activated cytotoxic T cells. There was no significant difference in CD4+ CD25+ cells between these HD groups, although the proportion of CD25+ cells within CD4+ cells increased in both groups as compared to normal controls. The serum titers of the thyroid autoantibodies and the proportion of CD25+ cells within CD8+ cells were higher in patients with severe HD than in those with mild HD. There was no correlation between these two parameters, and a two-dimensional analysis with these parameters differentiated these two groups of patients with HD more clearly. These results indicate that both thyroid autoantibodies and CD8+ CD25+ cells are independently involved in the disease severity of HD and CD4+ CD25+ cells are not related to the severity of HD.

HLA-A and -DRB4 genes in controlling the susceptibility to Hashimoto's thyroiditis

HLA-linked genetic factors involved in the pathogenesis of HT were studied in 71 patients with HT by serologic typing for HLA-A, -B, -C, -DR, and -DQ specificities and by DNA typing for HLA-DRB1, -DRB3, -DRB4, -DRB5, -DQA1, -DQB1, AND -DPB1 genes using the PCR-SSOP method. Typing results demonstrated significant positive associations of HT with HLA-A2 and -DRB4*0101 (DR53) (p < 0.01, RR = 2.03, EF = 0.61 and p < 0.0001, RR = 4.48, EF = 0.69, respectively). Although HLA-DR8, -DRB1*0403, -DQA*03, and -DQB1*0303 were statistically more prevalent in the patient group than in the controls, these associations were presumably due to the strong linkage disequilibria of these alleles with HLA-A2 or -DRB4*0101 in the Japanese population. Ninety-seven percent of the patients (69 out of 71) were positive for HLA-A2 or -DRB4*0101 compared to 79% in controls (RR = 8.7, p < 0.0005). The combination of HLA-A2 and -DRB4*0101 showed higher OR of risk for HT (OR = 12.8) than HLA-A2 (OR = 7.3) or DRB4*0101 (OR = 7.5) alone. These observations suggest that at least two loci, HLA-A and HLA-DRB4 together, may control the susceptibility to HT. On the other hand, the frequency of DQA1*0102 was significantly decreased in the patient group, suggesting that DQA1*0102 might confer resistance to HT.

Changes in plasma cholecystokinin concentrations after oral glucose tolerance test in anorexia nervosa before and after therapy.

There is considerable evidence that the gastrointestinal hormone cholecystokinin (CCK) induces satiety and reduces food intake in both animals and humans. Impaired CCK secretion was recently reported in patients with bulimia nervosa (BN) in whom plasma CCK responses to a standardized mixed-liquid meal were significantly lower than in controls. The present study was undertaken to determine whether CCK levels were abnormal in another relatively common eating disorder, anorexia nervosa (AN), before and after therapy and to investigate the relationship to the abnormal eating behavior. Plasma CCK, serum glucose, and immunoreactive insulin (IRI) responses to a 50-g oral glucose load were measured in 13 women with AN and in nine normal sex- and age-matched controls. The AN patients were all hospitalized during treatment; following partial restoration of body weight, the tests were repeated. Initial body weights were 70.8% +/- 1.8% (mean +/- SEM) of ideal body weight (IBW), and following partial restoration were 84.3% +/- 1.4%. Body weights in normal controls were 96.3% +/- 2.1% of IBW. Initial basal CCK concentrations in the AN patients before nutritional and cognitive behavioral therapy were significantly greater than those in controls (P < .01). After partial restoration of body weight, basal CCK concentration in AN patients approached that of control subjects. When AN patients were given a glucose load before therapy, the change in CCK response was diminished when compared with that of controls. However, CCK responses to the glucose load in AN patients following therapy were similar to those of controls.(ABSTRACT TRUNCATED AT 250 WORDS)

METHIMAZOLE‐INDUCED AGRANULOCYTOSIS IN JAPANESE PATIENTS WITH GRAVES’ DISEASE

We reviewed the records of approximately 7000 Japanese patients whose hyperthyroidism was treated with methimazole (MMI) alone. Four patients (Group I) developed agranulocytosis during a second course of MMI therapy and eight patients (Group II) during an initial course. Six patients (three in each group) received less than 30 mg MMI daily. Agranulocytosis occurred after more than 2 months of therapy (12 weeks‐1 year) in five patients. Seven patients were less than 40 years of age. One patient displayed a gradual protracted development of agranulocytosis. These results indicate that agranulocytosis after MMI may occur irrespective of dose, age, duration of treatment, and with a second exposure.

Gastrin-releasing peptide immunoreactivity in medullary thyroid carcinoma

Two cases of gastrin releasing peptide (GRP)‐producing medullary thyroid carcinoma are presented. Immunohistochemical examination revealed the presence of GRP‐like immunoreactivity (IR‐GRP) in the primary tumor tissues. High concentration of IR‐GRP was also demonstrated in extracts of the primary tumors by radioimmunologic means with use of a GRP‐specific antiserum. Chromatographic analysis showed that the immunoreactivity was composed of at least two molecular forms: one behaved as synthetic porcine GRP on Sephadex G‐50 gel filtration and the other as porcine GRP (14–27), a C‐terminal active fragment of GRP. The IR‐GRP was shown not to be attributed to bombesin‐like immunoreactivity. Substance P‐like immunoreactivity was not detected in the tumor tissues by either immunohistochemical or radioimmunologic means. This is, as far as the authors are aware, the first finding of IR‐GRP as an ectopic product in medullary carcinoma.

Small bowel transit time measured by hydrogen breath test in patients with anorexia nervosa

The gastrocecal transit time was measured in 10 patients suffering from anorexia nervosa,using a lactulose hydrogen breath test, and was compared with the orocecal transit time in 11 healthy controls. One of the 10 patients and one of the 11 controls were excluded from this study because of no discernible increase in hydrogen excretion. The transit time was significantly prolonged in patients with anorexia nervosa compared with controls (117 min ±31 sd vs 81 min + 33 SD, P <0.02). In addition to delayed gastric emptying, which has hitherto been well known, the small bowel transit time was considered to be prolonged in patients with anorexia nervosa. Both these abnormalities seem to contribute to the development of various gastrointestinal symptoms in patients with anorexia nervosa.

Serum concentrations of remnant‐like particles in hypothyroid patients before and after thyroxine replacement

objectives  Remnant‐like particles (RLPs) reflect chylomicron remnants and very‐low‐density lipoprotein remnants, which are most likely to be atherogenic particles. To investigate the effect of thyroxine replacement on the metabolism of RLPs in hypothyroidism, we measured serum concentrations of RLPs during an oral fat‐loading test in patients with hypothyroidism before and after thyroxine replacement.

Hashimoto's thyroiditis manifesting monoclonal lymphocytic infiltration

Hashimotos thyroiditis (HT) and lymphoma arc sometimes difficult to distinguish between. Moreover, lymphoma sometimes develops in a thyroid gland from pre‐existing HT. Open‐ or large‐needle biopsy usually distinguishes between them; the specimen may be examined histologically and subjected to immunohistochemistry. Another possible method of examination is fine‐needle aspiration biopsy (FNAB). The cells obtained may be evaluated cytologically, and subjected to flow cytometry, using various antibodies. In this study, anti‐kappa and anti‐lambda antibodies are especially important, as a gross predominance of kappa or lambda B lymphocytes infiltrating the thyroid is evidence for a B cell monoclone. In this study, 15 patients were selected because of their rapidly growing goitres. They all underwent FNAB. Five had cytology typical of HT, and no evidence of monoclonality on flow cytometry. They were diagnosed as HT without further histopathology. The remaining 10 patients had cytology suspected of lymphoma, or evidence of monoclonality on flow cytometry, or both. These patients underwent open‐ or large‐needle biopsy. Only three of them were diagnosed histopathologically as lymphoma; the other seven were diagnosed histopathologically as HT, making 12 cases of HT in all. Five of these 12 cases, and one of the three cases of lymphoma showed flow cytometrical evidence of monoclonality; thus evidence of monoclonality from FNAB, while interesting, does not necessarily serve to differentiate between HT and lymphoma. Furthermore, the immunohistochemical assessment of monoclonalily did not correlate with the flow cytometrical assessment. Follow‐up evidence will be required to discover whether those patients with a B cell monoclone in their HT are the ones who develop a lymphoma.