Insulin-dependent Diabetes Mellitus Patient Research Articles

Linkage of type I diabetes to 15q26 (IDDM3) in the Danish population.

Affected sib pair and linkage disequilibrium analysis, intrafamilial and case-control association studies were performed on 81 Danish multiplex insulin-dependent diabetes mellitus (IDDM) families (382 individuals) and 82 healthy Danish controls. The results confirm the linkage of D15S107 to IDDM in these Danish IDDM families (P = 0.010). When these data are combined with those of previous studies, an even stronger case for linkage can be made (P = 0.0005). Our analyses show that the D15S107*130 allele provides increased susceptibility (P = 0.02, relative risk = 3.55) and that the D15S107 locus contributes up to 16% of the familial clustering of IDDM. The analysis of affected sib pairs suggests that HLA and D15S107 may possibly act independently of each other. Taken together with our previous findings, our results suggest that three causes of susceptibilities can be discerned in the IDDM patient population: (1) a major susceptibility caused by the HLA-DRB1 alleles; (2) a minor susceptibility caused by the joint action of HLA and other non-HLA gene(s); and (3) a minor susceptibility caused by non-HLA gene(s).

In vitro stimulation with glutamic acid decarboxylase (GAD65) leads to an oligoclonal response of peripheral T-cells in an IDDM patient.

The enzyme glutamic acid decarboxylase (GAD65) is a major autoantigen in insulin-dependent diabetes mellitus (IDDM). To study T-cell reactivity towards GAD, peripheral blood leucocytes from seven patients with IDDM and five control subjects were stimulated in vitro with recombinant GAD. All diabetics studied were heterozygous for diabetes-associated HLA alleles, i.e. HLA-DRB1*03,*04-DQB1 *0302,*0201. A single IDDM subject (no. GAD65.05) revealed a strong response against GAD65. After stimulation, his T-cell receptor beta (TCRBV) usage was found to be oligoclonal. The sequence analysis of the putative peptide binding region of the T-cell receptor (CDR3 region) of 37 GAD-reactive T-cell clones revealed no common CDR3 motif. The stimulation of GAD-reactive T-cells could be inhibited with anti-class II monoclonal antibodies, indicating a class II restricted T-cell response. In addition, GAD65-responsive T-cells revealed a Th1 cytokine response pattern. The author's data suggest that GAD-reactive T-cells of Th1 phenotype can be obtained after in vitro stimulation of peripheral blood leucocytes from an HLA-DRB1*03/*04 heterozygous IDDM patient. The lack of a common CDR3 motif suggests the absence of an immunodominant T-cell epitope in that patient, or may indicate receptor repertoire spreading of peripheral T-lymphocytes.

The 37/40-kilodalton autoantigen in insulin-dependent diabetes mellitus is the putative tyrosine phosphatase IA-2.

Major targets for autoantibodies associated with the development of insulin-dependent diabetes mellitus (IDDM) include tryptic fragments with a molecular mass of 37 kDa and/or 40 kDa of a pancreatic islet cell antigen of unknown identity. The assay identifying autoantibodies against the 37/40-kDa antigen in human sera is based on the immunoprecipitation of 35S-labeled rat insulinoma cell proteins with sera from IDDM patients, followed by limited trypsin digestion of the immunoprecipitated material. To identify cDNA clones coding for the 37/40-kDa antigen, we have screened a cDNA expression library from rat insulinoma cells with a serum from an IDDM patient that precipitated the 37/40-kDa antigen in our assay. Among the cDNA products that reacted with the IDDM serum, we identified one cDNA clone whose open reading frame encodes a protein with a predicted mass of 105 kDa that we termed "ICA105" for 105-kDa islet cell antibody. The deduced amino acid sequence has high homology to a recently cloned putative tyrosine phosphatase IA-2 from human and mouse cDNA libraries. Translation of the cDNA in vitro results in a polypeptide with the expected molecular mass of 105 kDa. The evidence that ICA105 is indeed the precursor of the 37/40-kDa tryptic fragments is based on the following three results: (i) Sera from IDDM patients containing autoantibodies to the 37/40-kDa antigen precipitate the in vitro translated polypeptide, whereas sera from healthy subjects as well as sera from IDDM patients not reactive with the 37/40-kDa antigen do not precipitate the cDNA product. (ii) Immunoprecipitation of the in vitro translated protein with sera containing autoantibodies to the 37/40-kDa antigen followed by limited trypsin digestion of the precipitated proteins results in a 40-kDa polypeptide. (iii) The protein derived from our cDNA but not from an unrelated control cDNA clone can block immunoprecipitation of the 37/40-kDa antigen from a labeled rat insulinoma cell extract. The availability of the cloned 37/40-kDa antigen should facilitate the identification of individuals at risk of IDDM with increased accuracy. Furthermore, the identification of the 37/40-kDa antigen as the putative tyrosine phosphatase IA-2 is of relevance in elucidating the role of this antigen in the development of IDDM.

Usefulness of the American College of Rheumatology recommendations for liver biopsy in methotrexate-treated rheumatoid arthritis patients.

To test the usefulness and cost savings resulting from application of the new American College of Rheumatology (ACR) guidelines for assessing the risk for the development of clinically significant liver disease in rheumatoid arthritis (RA) patients treated with methotrexate (MTX). One-hundred twelve MTX-treated RA patients were prospectively followed up for MTX hepatotoxicity and underwent liver biopsies according to modified guidelines of the Psoriatic Task Force (PTF). All biopsies were graded according to the Roenigk classification. The new ACR recommendations were then retrospectively applied to test their usefulness and cost-effectiveness in this cohort. Based on the PTF guidelines, 66 patients underwent liver biopsies; a total of 110 liver biopsies were performed. Two patients had biopsy-related complications. Five patients were found to have Roenigk grade IIIB or IV histologic abnormalities. The total cost for this group was $111,380. Applying the new ACR criteria, only 15 patients would have undergone liver biopsies; there would have been a total of 18 biopsies, with no complications. Four of the 5 patients with Roenigk grade IIIB or IV liver abnormalities would have been identified. One patient with insulin-dependent diabetes mellitus (IDDM) who was found to have cirrhosis (Roenigk grade IV) on liver biopsy as a result of use of the PTF guidelines would have been missed with use of the ACR guidelines. The total cost for the group receiving biopsies based on the ACR guidelines would have been $16,956. Overall, the new ACR guidelines had 80% sensitivity and 82% specificity and resulted in a cost savings of $1,430 per patient. The new ACR guidelines on MTX monitoring and biopsy surveillance appear to be clinically useful and result in considerable cost savings. However, 1 IDDM patient with significant liver histologic abnormalities would have been missed. We suggest that IDDM be added to the ACR guidelines as a risk factor for MTX hepatotoxicity.

Association of polymorphism in the interferon gamma gene with IDDM.

Cytokines may play important roles in the pathogenesis of insulin-dependent diabetes mellitus (IDDM). We analysed a dinucleotide repeat polymorphism within the first intron of the interferon gamma (IFN-gamma) gene in Japanese diabetic patients (175 IDDM and 145 non-insulin-dependent diabetes mellitus) and 267 control subjects. A significant difference was observed in the global allele distribution of the polymorphism between the IDDM and control groups (p = 0.039). The difference from the control group was more evident in the patients whose insulin therapy started within 1 year from onset (p = 0.006) or in the young-onset (< 10 years) patients (p = 0.0006). The alleles "3" and "6" were increased in the IDDM patients, and a significant increase in the frequency of the "3/6" genotype was observed in the IDDM patient group (9.1%, RR 2.9, p = 0.010), in the patients with initial insulin therapy less than 1 year from onset (10.6%, RR 3.4, p = 0.004), or in the young-onset patients (16.7%, RR 5.7, p = 0.0003) in comparison to the control subjects (3.4%). There was a tendency towards frequent occurrence of clinical characteristics which reflect young or abrupt onset of diabetes or both, and depletion of insulin secretion capacity in the patients with "3/6" or "6/6" in comparison to the patients with other genotypes. These results suggest that the IFN-gamma gene region may contribute to the pathogenesis of IDDM and could be a genetic marker for IDDM.

The prevalence of coeliac disease in adult diabetes mellitus

Coeliac disease occurs more commonly in children with insulin-dependent diabetes mellitus (IDDM) than in the general population, but the prevalence of coeliac disease in adults with diabetes is unknown. We therefore screened an adult hospital-based diabetic population using IgA antigliadin antibody (IgA-AGA) to identify those patients requiring intestinal biopsy. In 1 year, 1789 patients (43% IDDM, 57% NIDDM) were screened, and 73 had raised IgA-AGA. Of these patients, 49 agreed to duodenal biopsy and 13 (10 IDDM) had coeliac disease. Selective IgA deficiency was found in eight patients, one of whom had coeliac disease. Of these 14 patients with newly diagnosed coeliac disease, four had microcytic anaemia, nine a low serum ferritin, and four a low albumin-corrected calcium. Eight patients had symptoms which improved on gluten withdrawal. Dietary compliance was maintained in 6/8 symptomatic patients, but only in 1/6 without symptoms. Included in the 1789 patients were four (all IDDM) with known coeliac disease. The overall prevalence of coeliac disease in adult patients with IDDM was 1:50 compared with 1:340 in NIDDM. Coeliac disease is common in adults with IDDM and may cause malabsorption and ill health. It should be suspected in any IDDM patient with gastrointestinal symptoms or unexplained anaemia.

An integral membrane protein form of brain l-glutamate decar☐ylase: purification, characterization and its relationship to insulin-dependent diabetes mellitus

A new and novel form of l-glutamate decar☐ylase (GAD; EC 4.1.1.15) was purified from whole porcine brain to apparent homogeneity by a combination of column chromatographies on DE-52, ultragel AcA 34, hydroxylapatite and Sephadex G0200, and native gel electrophoresis. The purified GAD was established as an integral membrane protein based on hydrophobic interaction chromatography and membrane extraction studies. This membrane GAD (MGAD) has a native molecular weight of 120 ± 5 kDa and is a homodimer of 60 ± 2 kDa. Immunoprecipitation and immunoblotting tests using the sera form insulin-dependent diabetes mellitus (IDDM) patient revealed the presence of antibodies against this newly identified MGAD in IDDM. The role of MGAD in the pathogenesis of IDDM and related autoimmune disorders is also discussed.

A novel radioligand binding assay to determine diagnostic accuracy of isoform-specific glutamic acid decarboxylase antibodies in childhood IDDM.

Insulin-dependent diabetes mellitus (IDDM) is associated with autoreactivity against GAD but the diagnostic sensitivity (positivity in disease) and specificity (negativity in health) of isoform-specific GAD antibodies have yet to be defined in assay systems suitable for screening large number of samples. One set of IDDM patient (n = 10) and control (n = 50) standard sera were used to develop quantitative antibody assays with in vitro synthesized recombinant 35S-methionine-labelled GAD65 and GAD67, respectively, and protein A-Sepharose to separate free from antibody-bound ligand. Binding levels were not normally distributed (p < 0.0001) and therefore, the diagnostic accuracy of GAD antibodies was analysed by the ROC plots in population-based, consecutively-diagnosed, recent onset, 0-14 year-old patients (n = 105), and matched, healthy control subjects (n = 157). The ROC plots showed that the diagnostic sensitivity of GAD65 antibodies was 77% and the specificity 92% compared with 8% and 98%, respectively for GAD67 antibodies. In the IDDM sera, GAD65 and GAD67 antibodies were concordant in 7% (6 of 81) and GAD65 antibodies and ICA in 89% (72 of 81) without a correlation between the autoantibody levels. Autoantibodies to recombinant human islet GAD65 are specific and sensitive markers for childhood IDDM in this immunoassay with in vitro synthesized 35S-methionine-labelled recombinant GAD.

Platelet Na+/H+ antiport activity in patients with insulin-dependent diabetes mellitus with and without diabetic nephropathy.

The incidence of diabetic nephropathy in patients with insulin-dependent diabetes mellitus (IDDM) may depend on factors other than the quality of diabetes control. Hypertension is an additional factor associated with a high degree of renal involvement in IDDM. One abnormality consistently observed in various tissues of patients with essential hypertension is enhanced activity of the Na+/H+ antiport. In the present study we have therefore studied platelet antiport activity in 41 healthy subjects (control), in 22 patients with untreated essential hypertension (EH), and in 35 normotensive IDDM patients (type 1). Of these patients 17 exhibited signs of diabetic nephropathy (group 1) while 18 had no evidence for renal involvement of IDDM in spite of a duration of IDDM of at least 10 years (group 2). The two IDDM patient groups were undistinguishable with respect to age, body mass index, and arterial blood pressure (group 1, 117.9 +/- 2.4/78.4 +/- 1.5 mmHg; group 2, 113.9 +/- 3.6/76.1 +/- 1.8 mmHg). Antiporter activity was determined from the rate of cell volume changes induced by propionic acid. Platelet Na+/H+ exchange activity averaged 23.43 +/- 0.43 10(-3).s-1 in control subjects and was markedly elevated in EH (28.38 +/- 0.62 10(-3).s-1; P < 0.01). Antiport activity in group 2 patients without nephropathy averaged 24.54 +/- 0.57 10(-3).s-1 and was undistinguishable from the control group. However, platelet Na+/H+ antiport activity was significantly stimulated in group 1 patients with nephropathy as compared to group 2 (26.95 +/- 0.73 10(-3). s-1; P < 0.025). Our results show that renal involvement in IDDM is associated with enhanced activity of the platelet Na+/H+ antiport.

Glucose regulation of the autoantigen GAD65 in human pancreatic islets.

Recent data suggest that the enzyme glutamic acid decarboxylase (GAD) may be a primary beta-cell antigen involved in the autoimmune response leading to insulin dependent diabetes mellitus (IDDM). Following three days culture of human islets at different glucose concentrations (5.6, 11 or 28 mM), there was a single 65 kDa/GAD band in the islet extracts, as assessed by immunoprecipitation both with serum from a newly diagnosed IDDM patient and a sheep anti-GAD serum. The synthesis of GAD was strongly stimulated at the higher glucose concentrations. Likewise, Western blot analysis indicated a glucose-induced increase of GAD in the islets. The data suggest that an enhanced function of human islet cells increases expression of GAD65. Possibly, this could exacerbate the autoimmune assault in the early stages of IDDM, and be of practical importance in attempts to ameliorate the autoimmune response towards the beta-cells in IDDM.

A 64 kDa antigen/glutamic acid decarboxylase (GAD) in fetal pig pro-islets: Co-precipitation with a 38 kDa protein and recognition by T cells in humans at risk for insulin-dependent diabetes

The development of insulin-dependent diabetes mellitus (IDDM) is associated with circulating antibodies to a pancreatic islet protein of MW 64,000 (64 kDa), reported to be glutamic acid decarboxylase (GAD). To investigate the antigenic properties of the 64 kDa antigen/GAD in IDDM we employed fetal pig pro-islets, a convenient source of islet antigens and an alternative to adult human islets for transplantation. Pro-islets contained a 64 kDa protein precipitated by antibodies in the sera of 9 14 (64%) at-risk IDDM, 12 33 (36%) recent-onset IDDM and 4 12 (33%) established IDDM patients and in 1 18 (5%) healthy control subjects. In addition, a 38 kDa protein was co-precipitated with the 64 kDa protein by 6 14 (43%) at-risk IDDM, 5 33 (12%) recent-onset IDDM, 1 12 (8%) established IDDM patient sera and 1 18 (5%) control subject serum. Both 64 kDa and 38 kDa antigens were specific to pro-islets; neither was detected in fetal pig thyrocytes, hepatocytes or splenocytes. The majority of the 64 kDa protein was co-precipitated with GAD enzymatic activity from pro-islets by either IDDM sera or a sheep anti-GAD serum. Previously, we showed that peripheral blood mononuclear cells (PBMC) from over half the subjects defined as being at risk for IDDM proliferate in response to fetal pig pro-islets. Proliferative responses of PBMC from pre-diabetic subjects to a crude extract of pro-islets were therefore measured before and after depletion of GAD by adsorption of the extract against GAD-1 monoclonal antibody. In 5 10 at-risk subjects, T cell stimulation indices were greater than the control mean+2 SD and decreased in four by > 50% after depletion of GAD. In summary, a 64 kDa protein with the properties of GAD is present in fetal pig pro-islets and is recognized by both antibodies and T cells in a significant proportion of subjects at risk for early IDDM. Some subjects with anti-64 kDa antibodies also have antibodies that co-precipitate a 38 kDa pro-islet protein. Prospective studies of T cell reactivity in at-risk IDDM subjects are required to delineate the role of GAD and other candidate autoantigens in the initiation and progression of β cell destruction.

Microbiology of healthy and diseased periodontal sites in poorly controlled insulin dependent diabetics.

A group of poorly-controlled insulin dependent diabetes mellitus (IDDM) patients were examined in a cross-sectional design for total microbial levels, microbial incidence, and the percent levels of selected periodontal microorganisms. These organisms were selected on the basis of prior reports that associated them with either periodontal disease or health. One periodontally-healthy and one periodontally-diseased site were examined in each IDDM patient. Increased levels of the periodontal pathogens Prevotella intermedia, P. melaninogenica spp., Bacteroides gracilis, Eikenella corrodens, Fusobacterium nucleatum and Campylobacter rectus (formerly Wolinella recta) were found at the periodontal diseased sites. Increased prevalence of the organisms P. intermedia, P. melaninogenica spp., and C. rectus were found at the diseased sites. A significantly higher percentage of P. intermedia was found at the sites exhibiting deep pockets and attachment loss.

Insulin-pen treatment, quality of life and metabolic control: retrospective intra-group evaluations

The significance of the insulin pen for the quality of life of patients with insulin-dependent diabetes mellitus (IDDM) has been debated. The aim of this study was to empirically investigate whether quality of life and metabolic control improve and whether insulin requirements change subsequent to multiple injection-pen treatment. The study group comprised 72 consecutive outpatients with IDDM. Thirty-eight subjects had an initial daily regimen of one or two injections, and the remaining 34 subjects had three or more injections. All patients had four or five injections per day during pen treatment. Perceived changes in quality of life attributed to pen treatment were assessed retrospectively at follow-up 9–13 months after the changeover. Data on metabolic control (HbA 1c) and insulin dose were collected at base-line and follow-up. The life quality of the IDDM-patients improved consistently, a finding corroborated by recent studies with other designs and methods. Metabolic control improved only for those patients who previously had one or two injections. The insulin requirements did not change. In conclusion, the pen contributes to a better life for the IDDM patient. The quality of life changes due to treatment intervention appear to be assessable.

Corneal autofluorescence in diabetic and penetrating keratoplasty patients as measured by fluorophotometry

At first it was verified that the major part of the fluorophotometer signal obtained when measuring corneal autofluorescence originated from fluorescence and not from scatter of excitation light at the corneal surface. The minimum percentage of the signal which can be attributed to fluorescence was determined using a fluorescence blocking filter placed in the excitation and fluorescence light path, respectively. The minimum percentages in two healthy controls, two diabetic patients and two patients after penetrating keratoplasty ranged from 75% to 93% (mean 84%). Then the corneal autofluorescence was determined in 22 healthy controls, 18 non-insulin-dependent diabetes mellitus (NIDDM), 23 insulin-dependent diabetes mellitus (IDDM) and 21 penetrating keratoplasty patients in order to detect a possible difference in autofluorescence as a result of diabetes or penetrating keratoplasty. The means of the corneal peak autofluorescence values in the NIDDM, IDDM and penetrating keratoplasty patient groups were significantly higher than that in the healthy control group (mean values in ng equivalent fluorescein ml −1: 18·0±4·2 s.d., 20·6±5·1 s.d., 17·9±5·5 s.d. and 13·7±3·7 s.d., respectively; P < 0·01). The mean values in the NIDDM and IDDM patients did not differ significantly ( P = 0·09). The autofluorescence values were independent of age in all four groups (linear correlation coefficient: r < 0·47). The corneal peak autofluorescence was linearly correlated with the diabetes duration in the NIDDM and IDDM patients [ r = 0·6, P = 0·02; increase: 0·36 ng equiv. fluorescein ml −1 yr diabetes −1]. Our results show that corneal autofluorescence is an easily obtained parameter which can be of assistance in evaluating corneal metabolism.

Morphotypes of the subgingival microflora in diabetic adolescents in Finland.

The morphotypes of the subgingival microflora from 85 12 to 18-year-old Finnish adolescents with insulin-dependent diabetes mellitus (IDDM) were studied in Gram- and Rhodes-stained smears. A comparison was made with subgingival plaque samples from paired age- and sex-matched healthy controls. Significant differences were found in the distribution of the morphotypes. The microflora in the IDDM patient group contained significantly lower proportions of Gram-positive and Gram-negative cocci and total Gram-positive bacteria and higher proportions of Gram-negative rods, fusiforms, and total Gram-negative bacteria. In the Rhodes-stained samples, the patients had more straight and curved rods and less fusiforms than the controls. The proportions of spirochetes and flagellated bacteria were almost identical in both groups. The clinical periodontal status of the subjects had been reported in a separate study. In spite of similar Plaque Index scores, the patients had more gingivitis than the controls. This finding may be explained by the distribution of morphotypes: more Gram-negative rods and total Gram-negative bacteria (periodontally more pathogenic forms) in the diabetic patients.

The relationship of inflammatory periodontal disease to diabetic status in insulin-dependent diabetes mellitus patients.

This study was designed to evaluate the relationship of inflammatory periodontal disease to the diabetic status of the insulin-dependent diabetes mellitus (IDDM) patient. 52 IDDM patients, ages 11-22 years, were evaluated. These patients were closely monitored at regular intervals in the University of Kentucky pediatric diabetic clinic. A periodontal examination was carried out for each patient. The patients were then assigned to a periodontitis or non-periodontitis group. Moderate to advanced periodontitis was found in 5.8% of the subjects. The gingival index and sulcular bleeding index were significantly higher in the periodontitis group (P less than 0.05). There was no significant difference between groups for plaque index, age of diabetic onset, duration of diabetes, present age, insulin dosage/weight, or serum glucose (P greater than 0.05). There was a greater % of ketoacidosis, retinopathy and neuropathy in the periodontitis group. IDDM patients with neurological complications or a history of chronic infections had a significantly higher gingival index score than those without the complication (P less than 0.05).

Sequence analysis of HLA class II genes from insulin-dependent diabetic individuals

To examine the nature of HLA-linked genetic susceptibility to insulin-dependent diabetes mellitus (IDDM), we compared HLA class II gene sequences from IDDM patients and control individuals. Genomic libraries were constructed from two siblings with IDDM, typed serologically as DR3,w6 and DR3,4. These libraries represent the HLA haplotypes (DR3, DR4) most frequently associated with IDDM, as well as one haplotype found less often. Individual genomic clones were identified and assigned to specific loci and haplotypes. The nucleotide sequence was then determined from the variable second exon from the HLA-DQα, DQß, and DRß genes from all three haplotypes. Sequence variation within the DQα genes could not be correlated with the disease. For all three haplotypes, the DQα sequence from the IDDM patient was identical to the DR-matched control sequence. Similarly, for the DR3 haplotype, the DQß sequences matched all control DR3 alleles. The DQß sequence from the DR4 haplotype was identical to the predominant DR4 allele (DQß 3.2) but differed at four amino acid residues from the other major DR4 DQß sequence (DQß3.1) found rarely among IDDM patients. Sequence analysis of the DQß gene from the DRw6 haplotype revealed a new allele that differed from the DQß allele from a control DR6 allele at two residues. The DRß genes from these three haplotypes also did not show any sequence features uniquely associated with IDDM, although the frequency of certain allelic variants in all three of these haplotypes may be altered in the IDDM population. A particular group of amino acids was found to be shared between the DRß-1 alleles from the DR4 and DRw6 haplotypes and may be involved in genetic susceptibility to IDDM.

Molecular cloning of a polymorphic DNA endonuclease fragment associates insulin-dependent diabetes mellitus with HLA-DQ.

A BamHI 3.7-kilobase (kb) fragment detected by an HLA-DQ beta-chain complementary DNA (cDNA) probe and negatively associated with insulin-dependent diabetes mellitus (IDDM) was cloned and sequenced to localize the polymorphism to BamHI sites in intervening sequences of an HLA-DQ beta-chain gene. A probe of the first intervening sequence (IVS 1) showed the BamHI 3.7-kb fragment in 6 of 17 HLA-DR3/4 controls but in 0 of 13 DR-identical IDDM patients. All IDDM patients (13 of 13) had BamHI fragments of 12 and 4 kb, detected in 9 of 17 controls (P less than 0.02). The simple restriction fragment length polymorphism pattern of the IVS 1 probe was exploited by comparing 113 IDDM patients with 177 healthy controls to show increased prevalences in IDDM of the 12-kb (P less than 0.0001) and 4-kb (P less than 0.0001) fragments. In IDDM patients younger than 20 yr at onset, 98% were 12- and/or 4-kb positive, compared with 63% of controls (P less than 0.0001), giving a relative risk of 91.8 for individuals with both fragments. The 12-kb fragment was linked to HLA-DR4, and the 4-kb fragment to HLA-DR3. Both serologic markers were split and a non-DR3/non-DR4 IDDM patient was 4-kb positive. HLA-DQ seems therefore closer, than HLA-DR, to an IDDM susceptibility gene.

Characterization of three HLA-DR beta genes isolated from an HLA-DR 3/4 insulin-dependent diabetic patient.

Three HLA-DR beta genes were isolated from a Swedish HLA-DR3/4 insulin-dependent diabetes mellitus (IDDM) patient and characterized by restriction endonuclease mapping and nucleotide sequence analysis. Two out of the three DNA sequences differed from those of published DR beta-chain sequences. A DR beta-gene probe prepared from exon 4 and flanking sequences was used in a Southern blot analysis of blood donors' DNA and DNA from HLA-DR3/4 IDDM patients and HLA-DR-matched healthy control subjects. This probe differentiated HLA-DR3/4 IDDM patients and HLA-DR-matched controls in the Scandinavian population but not in the North American Caucasoid population.

A human monoclonal antibody to insulin.

To elucidate the immune aspects of insulin-dependent diabetes mellitus (IDDM), we attempted to generate human monoclonal anti-insulin antibodies by fusing peripheral blood lymphocytes obtained from 10 insulin-treated IDDM patients with cells from a human lymphoblastoid cell line. Hybridomas that secreted immunoglobulins appeared in 9 of 400 wells. One of these hybridomas secreted anti-insulin antibody of the IgM class. The lymphocytic partner of this hybridoma was obtained from an IDDM patient who had undetectable levels of antibodies to insulin in his serum. Thus, by employing the hybridoma technique, it was possible to reveal the presence of insulin-sensitized B-lymphocytes in a patient who was serologically negative for anti-insulin antibodies. The monoclonal antibody recognized intact human insulin and insulins of other species, but not isolated A- and B-chains. This indicates that the antibody was functionally an autoantibody directed to an epitope formed by the native conformation of a highly conserved portion of the insulin molecule. This is the first report of a human hybridoma antibody to insulin.