胰岛自身抗体对Ⅰ型糖尿病诊断效率的研究

目的 探讨免疫指标谷氨酸脱羧酶抗体（ＧＡＤ６５－Ａｂ）与胰岛细胞抗体（ＩＣＡ）对Ⅰ型糖尿病的诊断价值。方法 Ⅰ型糖尿病１０４例,正常对照１０２例,用放射配体分析法检测ＧＡＤ６５－Ａｂ,ＥＬＩＳＡ法检测ＩＣＡ;用受试者运筹特性（ＲＯＣ）曲线及曲线下面积比较二者的诊断效率。结果 （１）ＧＡＤ６５－Ａｂ最佳界值０．３０,ＩＣＡ最佳界值０．４５。ＧＡＤ６５－Ａｂ、ＩＣＡ、ＲＯＣ曲线下面积分别为０．８３５、     

人重组谷氨酸脱羧酶65基因的克隆表达及其在1型糖尿病诊断中的初步应用

目的 构建人重组谷氨酸脱羧酶65(GAD65)基因不同区段原核表达载体,诱导表达获得重组蛋白,并初步验证GAD65不同抗原区段在1型糖尿病GAD自身抗体检测中的价值.方法 应用巢式逆转录聚合酶链式反应(RT-PCR)技术调取目的 基因,构建相应的原核表达质粒,转化大肠杆菌E.coli HB101,诱导表达获得纯化重组蛋白,用重组蛋白作为包被抗原,初步建立检测GAD自身抗体的酶联免疫吸附测定法(ELISA)方法,评价各片段在1型糖尿病诊断中的价值.结果 获得了4种可被1型糖尿病患者血清识别的重组人GAD抗原区段,其中GAD65(180-585)抗原区段具有很好的特异性,检出率为55.3%,是首选的抗原区段.结论 所选重组人GAD65(180-585)抗原区段具有良好的抗原性,可作为1型糖尿病患者辅助诊断试剂的候选抗原.     

Enterovirus-related type 1 diabetes mellitus and antibodies to glutamic acid decarboxylase in Japan

OBJECTIVES: In order to clarify the relationship between enteroviruses and type 1 diabetes mellitus in Japan we investigated enteroviral RNA in serum from children with type 1 diabetes mellitus. METHODS: We investigated enteroviral RNA in serum from children with type 1 diabetes mellitus by using highly sensitive RT-PCR. Additionally the sequences and viral loads were determined and compared with anti-coxsackie virus antibodies and anti-glutamic acid decarboxylase (GAD) antibodies. RESULTS: RT-PCR for enterovirus was positive in 23 (37.7%) from 61 samples. The positivity had no disparity of age, but decreased by aging after the occurrence of type 1 diabetes mellitus. The sequences of the positives were similar as those of coxsackie B4. The viral loads revealed that there was no positive patient with high titers of anti-GAD antibodies. CONCLUSION: In Japan there is some correlation with type 1 diabetes mellitus and enterovirus. The pathophysiology of type 1 diabetes mellitus seems to consist of a direct destruction by persistent coxsackie virus and the autoimmune mechanism through autoantibodies against beta-cells.     

成人糖尿病患者血清GADA和ICA检测的临床意义

目的　研究血清胰岛细胞抗体 (ICA)、谷氨酸脱羧酶抗体 (GADA)检测对 1型糖尿病 (DM)早期诊断的意义 ,以及抗体阳性患者胰岛 β细胞功能。方法　采用 EL ISA法检测 ICA、GADA;放射免疫法检测血清空腹和餐后 C肽。结果　 16 3例 2型 DM患者中 ,ICA及 GADA阳性共 32例为 1型 DM,其中 ICA阳性 18例 ,GADA阳性 2 5例 ,GADA阳性率 (15 .3% )高于 ICA(11.0 % )。抗体阳性患者的空腹和餐后 C肽 [(0 .6 3± 0 .31)和 (1.76± 0 .82 ) pmol/ L]明显低于抗体阴性患者 [(0 .91± 0 .81)和 (3.18± 1.92 ) pm ol/ L]。GADA阳性患者的空腹和餐后 C肽 [(0 .2 9± 0 .18)和 (0 .74± 0 .4 3) pm ol/ L ]明显低于 ICA阴性患者 [(0 .4 8± 0 .32 )和 (1.11± 0 .4 5 )pmol/ L ]。结论　 ICA、GADA检测对 1型 DM的早期诊断有重要意义。     

Glutamic acid decarboxylase autoantibodies in preclinical insulin-dependent diabetes.

Insulin-dependent diabetes mellitus (IDDM) is associated with serum antibodies that precipitate a 64-kDa pancreatic islet cell protein reported to be glutamic acid decarboxylase (GAD; glutamate decarboxylase, EC 4.1.1.15). Previously, antibodies to GAD were found in the rare neurological disorder stiff man syndrome. To demonstrate directly antibodies to GAD, enzymatically active GAD was first purified from fresh human cerebellum. Brain GAD activity was precipitated by noninhibitory antibodies in the sera of 16/26 (62%) subjects defined as having preclinical IDDM (islet cell antibody-positive first-degree relatives of a person with IDDM), 3/13 (23%) with recent-onset IDDM, and 3/3 with the stiff man syndrome. In addition, sera of 5/26 (19%) preclinical and 2/13 (15%) recent-onset IDDM subjects contained antibodies that precipitated GAD but inhibited its activity. Thus, overall, 21/26 (81%) preclinical and 5/13 (38%) recent-onset IDDM subjects had antibodies that precipitated GAD activity. Antibodies to GAD were not detected in sera from subjects with other autoimmune diseases (n = 29) or healthy controls (n = 14). GAD affinity-purified to homogeneity (specific activity, 58 units/mg) was specifically immunoprecipitated as a single 60-kDa species by the IDDM sera. In an ELISA incorporating whole mouse brain GAD captured by the GAD-6 monoclonal antibody the frequencies of GAD antibodies for all subject groups were indistinguishable from those found by precipitation of human brain enzymatic activity. We conclude that (i) GAD is an (auto)antigen in a majority of subjects operationally defined as having preclinical IDDM, (ii) pancreatic islet and brain GAD are likely to be cross-reactive, and (iii) the majority of GAD antibodies are directed away from the catalytic site of the brain enzyme. The lower frequency of GAD antibodies in recent-onset IDDM subjects indicates either that immunoreactivity is lost with near-total beta-cell destruction or that GAD antibodies denote a low risk of progression to clinical disease.     

Ia-Type alloantigens and humoral autoimmune responsiveness in insulin-dependent diabetes mellitus.

In the search for markers either closely linked to or identical with the hypothetical "diabetogenic major histocompatibility gene," immune region-associated alloantigens were defined in 80 patients with insulin-dependent diabetes and in 107 controls. A close association between the Ia-type alloantigen DRw3 and DRw4 and insulin-dependent diabetes was obtained. DRw3 was found in 36% and DRw4 in 32% of the patients compared to 11% and 16%, respectively, of the controls. In addition, a significant influence of DRw3 and humoral anti-islet-cell autoimmunity could be observed, which was found to be due to a high incidence of DRw3 in those patients with islet cell antibody persistence. Islet-cell antibodies (ICA) were observed in 60% of the DRw3-positive patients compared to only 9% of the DRw3-negatives with longstanding disease (greater than 5 yr). These data show a significant association between insulin-dependent diabetes and the Ia-type alloantigens DRw3 (p uncorr. less than 0.0005) and DRw4 (p uncorr. less than 0.025). Furthermore, they provide direct evidence of an association between an Ia-type alloantigen and persisting humoral autoimmune responsiveness in man.     

Antibodies to glutamic acid decarboxylase are associated with HLA-DR genotypes in both Australians and Asians with Type 1 (insulin-dependent) diabetes mellitus

Summary Antibodies to glutamic acid decarboxylase, previously known as the 64 kD antigen, appear to be more predictive of Type 1 (insulin-dependent) diabetes mellitus in Caucasoids than other autoantibodies to islet cell antigens. However, seropositivity to glutamic acid decarboxylase is not universal at the onset of Type 1 diabetes and the prevalence in Asians is low compared to Caucasoid patients. This suggests the involvement of multiple pancreatic autoantigens in the Type 1 diabetes autoimmune process or, genetic differences within and between ethnic groups that contribute to the heterogeneous autoimmune response to glutamic acid decarboxylase or both. Alternatively some cases of Type 1 diabetes could have an aetiology unrelated to autoimmunity. This study examined the differential response to glutamic acid decarboxylase according to HLA-DR and -DQ genotypes, as determined by RFLP, in 49 white Australian and 44 Asian patients with Type 1 diabetes. Among Australians heterozygous for HLA-DR3, DR4, 85% were positive for antibodies to glutamic acid decarboxylase, significantly different (p = 0.039) from the prevalence of 48% in patients with at least one HLA-DR antigen other than DR3 or DR4. Also, among Australians, the presence of low risk HLA-DQ antigens, namely DQw5, DQw6 or DQw7, reduced the prevalence of antibodies to glutamic acid decarboxylase by 40% (p = 0.064). Among Asians with Type 1 diabetes and with antibodies to glutamic acid decarboxylase, HLA-DR9 was significantly (p = 0.037) increased in frequency, at 63% compared with 22% in those without glutamic acid decarboxylase antibodies, and the presence of a low risk HLA-DQ allele reduced the antibody rates by 87% (p = 0.003). These observations may reflect differential genetic/environmental interactions in Type 1 diabetes or differential persistence of glutamic acid decarboxylase antibodies in those with different genetic backgrounds.     

Development of autoimmune diabetes in glutamic acid decarboxylase 65 (GAD65) knockout NOD mice

Aims/hypothesis Type 1 diabetes mellitus, a T-cell-mediated autoimmune disease, results from the selective destruction of insulin-producing pancreatic beta cells. Autoantibodies against beta-cell components are used clinically as sensitive markers of this disease; however, their physiological role has not been clear. To investigate the role of glutamic acid decarboxylase 65 (GAD65) in the development of the Type 1 diabetes of non-obese diabetic (NOD) mice, we analysed and characterised NOD mice with targeted disruption of the GAD65 gene.Methods GAD65-deficient mice were previously established. After backcrossing the knockout mutation onto the NOD genetic background for up to eight generations, female littermates of the three resulting genotypes were produced by intercrossing: GAD65 +/+ (n=23), GAD65 +/– (n=62), and GAD65 –/– (n=31).Results The cumulative incidence of autoimmune diabetes showed no significant difference among the three groups in longitudinal studies using the Kaplan-Meier method. Islet morphology showed that the progression of islet infiltration did not differ significantly between the three groups.Conclusion/interpretation The cumulative incidence of autoimmune diabetes was not influenced by the GAD65 deficiency. These data suggest that GAD65 is not a major regulatory target of beta-cell autoimmunity in NOD mice.Abbreviations NOD Non-obese diabetic     

Prevention of autoimmune diabetes by immunogene therapy using recombinant vaccinia virus expressing glutamic acid decarboxylase

AIMS/HYPOTHESES: Type I (insulin-dependent) diabetes mellitus results from T-cell-mediated autoimmune destruction of pancreatic beta cells. Among the beta-cell autoantigens that have been implicated in triggering of beta-cell-specific autoimmunity, glutamic acid decarboxylase (GAD) is a strong candidate in both humans and the NOD mouse. We aimed to determine whether treatment with a recombinant vaccinia virus expressing GAD (rVV-GAD65) could prevent the development of diabetes in NOD mice. METHODS: Three-eight-to-nine-week-old female NOD mice were injected with various doses of rVV-GAD65 or rVV-MJ601as a control. We then examined the incidence of diabetes, T-cell proliferative response to GAD, amounts of anti-GAD IgGs, cytokine production and generation of regulatory cell populations. RESULTS: Administration of rVV-GAD65 to NOD mice prevented diabetes in an age-dependent and dose-dependent manner. Splenic T cells from rVV-GAD65-treated mice did not proliferate in response to GAD65. The amount of IgG1 was increased, whereas IgG2a amounts did not change in rVV-GAD65-treated NOD mice. The production of interleukin-4 increased, whereas the production of interferon-gamma decreased in rVV-GAD65-treated mice after stimulation with GAD. Furthermore, splenocytes from rVV-GAD65-treated NOD mice prevented the transfer of diabetes by splenocytes from acutely diabetic NOD mice in NOD. scid recipients. CONCLUSION/INTERPRETATION: Immunogene therapy using a recombinant vaccinia virus expressing GAD results in the prevention of autoimmune diabetes in NOD mice by the induction of immunological tolerance through active suppression of effector T cells, and this treatment might have therapeutic value for the prevention of Type I diabetes.     

HLA class II is associated with the frequency of glutamic acid decarboxylase M r 65 000 autoantibodies in Japanese patients with insulin-dependent diabetes mellitus

Autoantibodies to glutamic acid decarboxylase (GAD65Ab) are common in both caucasian and Japanese patients with insulin-dependent diabetes mellitus (type 1), while the type 1-associated HLA haplotypes differ. In the present study, we analyzed GAD65Ab in relation to HLA-DQ and-DR alleles in Japanese type 1 patients. GAD65Ab were found in 58% short-duration (less than 5 years) type 1,23% long-duration type 1,56% slowly progressive type 1,3% type 2 patients, and 1.7% healthy individuals. In 75 HLA-typed type 1 patients, the GAD65Ab frequency was higher in short-duration patients with DRB1^*08 allele (100%,Pc<0.05). GAD65Ab frequencies in DQB1^*0302, DQB1^*0303, and DRB1^*09-positive, long-duration type 1 patients were lower than those in short-duration type 1 patients (14%, 19%, and 20%,Pc<0.02 compared with short-duration type 1, 90%, 75%, and 71%, respectively), while the frequency varied less in DQB1^*04 individuals (44% and 30% in short- and long-duration type 1 patients, respecitively). These findings were also observed among patients with DRB1^*04, i.e., the haplotype DRB1^*0405-DQB1^*0401 showed less variation in frequency of GAD65Ab (44% and 35% in short- and long-duration type 1 patients, respectively), while DRB1^*04xx-DQB1^*0302 showed lower frequency in long-duration type 1 than short-duration (13% and 100%, respectively). Thus, HLA class II is associated with frequency GAD65Ab, and this association might be affected by disease duration in Japanese type 1 patients.     

Distribution of autoantibodies to glutamic acid decarboxylase across the spectrum of diabetes mellitus seen in South Africa.

AIMS: This study investigated the association between glutamic acid decarboxylase antibodies (GAD-AB) and Type 1, Type 2, pancreatic and lipoatrophic diabetes mellitus (DM) in South African patients. METHODS: Four groups were selected: group A, 100 Black Type 1 DM patients (age at onset < 35 years, body mass index (BMI) < 27 kg/m2 and insulin dependent within 1 year of presentation); group B, 80 Black Type 2 DM patients (age at onset > 35 years, BMI > 27 kg/m2 and controlled on oral hypoglycaemic agents for at least 1 year after presentation); group C, 10 patients of varying ethnicity with DM or impaired glucose tolerance secondary to chronic pancreatitis; group D, five patients of varying ethnicity with DM associated with total lipodystrophy. Fifty healthy Black control subjects were also studied (group E). Serum GAD-AB and random C-peptide levels were measured by radioimmunoassay. RESULTS: Mean C-peptide concentration was significantly lower in Type 1 DM patients than Type 2 DM patients (P < 0.00001). Forty-four patients with Type 1 DM were GAD-AB-positive compared to two patients with Type 2 DM. Two control subjects were also GAD-AB-positive. No patient in the other groups had a titre > 1 U/ml. Type 1 DM patients who were GAD-AB-positive did not differ from those who were GAD-AB-negative for age at onset, duration of DM or C-peptide concentrations. CONCLUSIONS: Auto-immune beta-cell destruction has an important role in the pathogenesis of Type 1 DM amongst African patients. However, Type 2 African DM patients and other diabetes subtypes are largely GAD-AB-negative.     

Target antigen of islet cell antibody in Japanese insulin-dependent diabetes mellitus

The target antigens of islet cell antibody (ICA) have not been clarified. We tried to modify the antigen in human pancreatic tissues and characterize the ICA with immunohistochemical methods. Human pancreatic tissues were treated with periodate (A), borohydride (B), neuraminidase (C), methanol (D), chloroform-methanol (E), or protease (F) to modify the antigens, and stained by an immunofluorescent method using ICA-positive sera from five Japanese insulin-dependent diabetes mellitus (IDDM) patients. In all sera the fluorescence of islets disappeared or waned after A, C, D, and E, and did not change after F. The disappearance or loss of fluorescence induced by A was recovered after B. It is, therefore, suggested that one of the antigens of ICA in Japanese IDDM patients is the sialic acid residue of glycolipid.     

Human monoclonal islet cell antibodies from a patient with insulin-dependent diabetes mellitus reveal glutamate decarboxylase as the target antigen.

The autoimmune phenomena associated with destruction of the beta cell in pancreatic islets and development of type 1 (insulin-dependent) diabetes mellitus (IDDM) include circulating islet cell antibodies. We have immortalized peripheral blood lymphocytes from prediabetic individuals and patients with newly diagnosed IDDM by Epstein-Barr virus transformation. IgG-positive cells were selected by anti-human IgG-coupled magnetic beads and expanded in cell culture. Supernatants were screened for cytoplasmic islet cell antibodies using the conventional indirect immunofluorescence test on cryostat sections of human pancreas. Six islet cell-specific B-cell lines, originating from a patient with newly diagnosed IDDM, could be stabilized on a monoclonal level. All six monoclonal islet cell antibodies (MICA 1-6) were of the IgG class. None of the MICA reacted with human thyroid, adrenal gland, anterior pituitary, liver, lung, stomach, and intestine tissues but all six reacted with pancreatic islets of different mammalian species and, in addition, with neurons of rat cerebellar cortex. MICA 1-6 were shown to recognize four distinct antigenic epitopes in islets. Islet cell antibody-positive diabetic sera but not normal human sera blocked the binding of the monoclonal antibodies to their target epitopes. Immunoprecipitation of 35S-labeled human islet cell extracts revealed that a protein of identical size to the enzyme glutamate decarboxylase (EC 4.1.1.15) was a target of all MICA. Furthermore, antigen immunotrapped by the MICA from brain homogenates showed glutamate decarboxylase enzyme activity. MICA 1-6 therefore reveal glutamate decarboxylase as the predominant target antigen of cytoplasmic islet cell autoantibodies in a patient with newly diagnosed IDDM.     

Subtle hyperproinsulinaemia characterises the defective insulin secretory capacity in offspring of glutamic acid decarboxylase antibody-positive patients with latent autoimmune diabetes mellitus in adults

OBJECTIVE: We set out to assess whether hyperproinsulinaemia is an early finding in latent autoimmune diabetes in adults (LADA). RESEARCH DESIGN AND METHODS: We measured plasma proinsulin and C-peptide responses during a 2-h oral glucose tolerance test (OGTT) and in the hyperglycaemic clamp in 21 normoglycaemic offspring of LADA patients testing positive for glutamic acid decarboxylase antibodies (GADA) or islet cell antibodies (ICA), and in 17 healthy control subjects without a family history of diabetes. RESULTS: The study groups had comparable areas under the curves of blood glucose, plasma proinsulin, C-peptide and proinsulin/C-peptide in the OGTT. However, the offspring of LADA patients had higher proinsulin/C-peptide in the hyperglycaemic clamp (P < 0.01 versus the control group). The offspring of GADA-positive LADA patients (n = 9) had higher proinsulin and proinsulin/C-peptide than did the control group in the OGTT (P < 0.05 for both comparisons) and in the hyperglycaemic clamp (P < 0.001 and P < 0.05 respectively). They also had higher proinsulin than the offspring of ICA-positive LADA patients (n = 12) (P < 0.001) in the hyperglycaemic clamp. The offspring of ICA-positive LADA patients did not clearly show hyperproinsulinaemia during the tests, but they had lower maximal glucose-stimulated insulin secretory capacity than the control group (P < 0.05) and the offspring of GADA-positive LADA patients (P < 0.05) in the hyperglycaemic clamp. CONCLUSIONS: These results suggested that insulin secretion in the offspring of GADA-positive LADA patients is characterised by subtle defects in the processing of insulin precursors. Furthermore, various proinsulin responses among the offspring of LADA patients with different autoimmune markers provided further evidence that LADA is a heterogeneous disorder.