Recent-onset Insulin-dependent Diabetes Mellitus Patients Research Articles

CTLA-4 gene polymorphism confers susceptibility to insulin-dependent diabetes mellitus (IDDM) independently from age and from other genetic or immune disease markers

SUMMARY Apart from genes in the HLA complex (IDDM1) and the variable number of tandem repeats in the 5′ region of the insulin gene (INS VNTR, IDDM2), several other loci have been proposed to contribute to IDDM susceptibility. Recently, linkage and association have been shown between the cytotoxic T lymphocyte-associated protein 4 (CTLA-4) gene on chromosome 2q and IDDM. In a registry-based group of 525 recent-onset IDDM patients < 40 years old we investigated the possible interactions of a CTLA-4 gene A-to-G transition polymorphism with age at clinical disease onset and with the presence or absence of established genetic (HLA-DQ, INS VNTR) and immune disease markers (autoantibodies against islet cell cytoplasm (ICA); insulin (IAA); glutamate decarboxylase (GAD65-Ab); IA-2 protein tyrosine phosphatase (IA-2-Ab)) determined within the first week of insulin treatment. In new-onset IDDM patients, G-allele-containing CTLA-4 genotypes (relative risk (RR) = 1.5; 95% confidence interval (CI) = 1.2–2.0; P < 0.005) were not preferentially associated with age at clinical presentation or with the presence of other genetic (HLA-DR3 or DR4 alleles; HLA-DQA1*0301-DQB1*0302 and/or DQA1*0501-DQB1*0201 risk haplotypes; INS VNTR I/I risk genotype) or immune (ICA, IAA, IA-2-Ab, GAD65-Ab) markers of diabetes. For 151 patients, thyrogastric autoantibodies (anti-thyroid peroxidase, anti-thyroid-stimulating hormone (TSH) receptor, anti-parietal cell, anti-intrinsic factor) were determined, but association between CTLA-4 risk genotypes and markers of polyendocrine autoimmunity could not be demonstrated before or after stratification for HLA- or INS-linked risk. In conclusion, the presence of a G-containing CTLA-4 genotype confers a moderate but significant RR for IDDM that is independent of age and genetic or immune disease markers.

Altered Peptide Ligands of Islet Autoantigen Imogen 38 Inhibit Antigen Specific T Cell Reactivity in Human Type-1 Diabetes

Type 1 diabetes, insulin-dependent diabetes mellitus (IDDM) results from autoimmune T cell-dependent destruction of insulin producing β-cells in the pancreatic islets of Langerhans. T cells from recent-onset IDDM patients specifically proliferate to β cell membrane Ag enriched fractions, containing the mitochondrial 38kD islet antigen (Imogen). Recently, we identified a peptide epitope (Imogen p55-70) that is recognized by a 38kD-specific, Th1 clone from an IDDM patient. In animal models of autoimmune diseases, altered self peptide ligands (APL) have been used effectively in peptide-based immune prevention or therapy. No such APL, however, have been reported so far that can modulate autoreactive T-cell responses in IDDM. Here, we have designed APL of p55-70. These APL efficiently downregulatein vitroactivation of the 38kD-specific Th1 clone induced by either p55-70 or by native β cell autoantigens. Self peptide reactive T-cell proliferation could be inhibited only when APL and the self peptide were present on the same APC. Unrelated peptides with equal HLA-DR binding affinity were not effective, excluding simple MHC competition as the mechanism for T-cell modulation. APL triggered upregulation of CD69 and CD25 expression, but not T-cell proliferation, TCR down-modulation or T-cell anergy. Thus, the p55-70 APL inhibit β cell autoantigen-induced activation of an Imogen-reactive T-cell clone derived from an IDDM patient, by acting as partial TCR agonists that inhibit TCR down-modulation.

Antibodies to the Protein Tyrosine Phosphatases IAR and IA-2 are Associated with Progression to Insulin-Dependent Diabetes (IDDM) in First-Degree Relatives At-Risk for IDDM

Insulin-dependent diabetes mellitus (IDDM) is preceded by the presence of antibodies against islet proteins including a protein tyrosine phosphatase (PTP) designated IA-2. Recently, we cloned a novel PTP named IAR which shares 43% sequence identity with IA-2 and is recognised by antibodies from a majority of patients with IDDM. The aim of the present study was to determine whether IAR antibodies (IAR Ab) or IA-2 antibodies (IA-2 Ab) are associated with progression to IDDM in first-degree relatives “at-risk” for IDDM (operationally defined as those with islet cell antibodies [ICA] ⩾ 20JDFU or insulin autoantibodies [IAA]> 100nU/ml), and to examine combinations of IAR Ab and IA-2 Ab in these subjects. The sensitivity and specificity of these antibodies were also examined in patients with recent-onset IDDM. Using Cox's Proportional Hazards Model, the number of siblings with IDDM was associated with progression to IDDM in “at-risk” relatives, but other covariables (age, sex, number of affected offspring or parents) were not significantly associated. Using number of affected siblings as a covariable, both IAR and IA-2 antibodies were significantly associated with progression to IDDM (p < 0.005). Combinations of both antibodies, however, did not result in a significantly stronger association with progression to IDDM. The threshold of positivity for IAR Ab (0.5 units) and IA-2 Ab (3.0 units) assays was adjusted to give the same specificity (97.9%) for each assay in 144 healthy control subjects, to allow standardised comparisons. Levels of IAR Ab and IA-2 Ab were strongly correlated in 53 recent-onset IDDM patients (r = 0.70, p< 0.0001) but 11.3% had IAR Ab in the absence of IA-2 Ab and 16.9% had IA-2 Ab in the absence of IAR Ab. The sensitivity for IDDM (defined as the proportion of IDDM patients positive) was 56.6% for IAR Ab and 62.3%, for IA-2 Ab. We conclude that there is considerable overlap in IA-2 Ab and IAR Ab positivity, although either antibody can occur independently in IDDM patients. Both IAR Ab and IA-2 antibodies are associated with progression to IDDM in first-degree relatives at-risk of IDDM, but the use of IAR and IA-2 antibodies in combination are not significantly more strongly associated with progression than single antibodies. IAR Ab may play an important role in the prediction of IDDM.

Increased prevalence of abnormal immunoglobulin M, G, and A concentrations at clinical onset of insulin-dependent diabetes mellitus: a registry-based study. The Belgian Diabetes Registry.

In view of the reported association of insulin-dependent diabetes mellitus (IDDM) with viral infections, (non-) islet-specific immune changes, and partial immunodeficiencies, we used immunonephelometry to measure circulating levels of IgM, IgG, and IgA in a registry-based group of IDDM patients under age 40 years at clinical onset (n = 397) and in age-matched nondiabetic siblings (n = 316) and control subjects (n = 322). Overall, IgM and IgA concentrations were higher, and IgG concentrations lower, in patients than in control subjects or siblings (P < 0.001). In siblings, IgM concentrations were higher than in control subjects (P < 0.001). Using age-adjusted reference intervals, abnormal Ig concentrations were noted in 27% of the patients at onset versus only 10% of the siblings and 7% of the control subjects (P < 0.001). For onset between 20 and 40 years (n = 220), 30% of the patients presented either increased IgM levels (21%) or decreased IgG levels (11%). Both independent phenomena occurred regardless of diabetes-associated immune and genetic markers. In patients and siblings, IgM levels were positively correlated with pancreatic lipase activity. In diabetic children (0-9 years; n = 65), a subgroup presented increased IgA levels (15%) in association with the highest HLA DQ-linked genetic risk and elevated IgM levels. The changes in total Ig concentrations at onset were largely reversed under insulin therapy. They may reflect exposure to environmental triggers, such as viral infections, or to (relative) insulinopenia prior to clinical disease onset. Whatever their cause, different serum Ig levels exist in different age groups of recent-onset IDDM patients.

CTLA-4 gene polymorphism confers susceptibility to insulin-dependent diabetes mellitus (IDDM) independently from age and from other genetic or immune disease markers. The Belgian Diabetes Registry.

Apart from genes in the HLA complex (IDDM1) and the variable number of tandem repeats in the 5' region of the insulin gene (INS VNTR, IDDM2), several other loci have been proposed to contribute to IDDM susceptibility. Recently, linkage and association have been shown between the cytotoxic T lymphocyte-associated protein 4 (CTLA-4) gene on chromosome 2q and IDDM. In a registry-based group of 525 recent-onset IDDM patients <40 years old we investigated the possible interactions of a CTLA-4 gene A-to-G transition polymorphism with age at clinical disease onset and with the presence or absence of established genetic (HLA-DQ, INS VNTR) and immune disease markers (autoantibodies against islet cell cytoplasm (ICA); insulin (IAA); glutamate decarboxylase (GAD65-Ab); IA-2 protein tyrosine phosphatase (IA-2-Ab)) determined within the first week of insulin treatment. In new-onset IDDM patients. G-allele-containing CTLA-4 genotypes (relative risk (RR)= 1.5; 95% confidence interval (CI) = 1.2-2.0; P < 0.005) were not preferentially associated with age at clinical presentation or with the presence of other genetic (HLA-DR3 or DR4 alleles; HLA-DQA1*0301-DQB1*0302 and/or DQA1*0501-DQB1*0201 risk haplotypes; INS VNTR I/I risk genotype) or immune (ICA, IAA, IA-2-Ab, GAD65-Ab) markers of diabetes. For 151 patients, thyrogastric autoantibodies (anti-thyroid peroxidase, anti-thyroid-stimulating hormone (TSH) receptor, anti-parietal cell, anti-intrinsic factor) were determined, but association between CTLA-4 risk genotypes and markers of polyendocrine autoimmunity could not be demonstrated before or after stratification for HLA- or INS-linked risk. In conclusion, the presence of a G-containing CTLA-4 genotype confers a moderate but significant RR for IDDM that is independent of age and genetic or immune disease markers.

Identification of naturally processed T cell epitopes from glutamic acid decarboxylase presented in the context of HLA-DR alleles by T lymphocytes of recent onset IDDM patients.

Glutamic acid decarboxylase (GAD) has been defined as a major target antigen in insulin-dependent diabetes mellitus (IDDM). To identify the molecular ligands triggering a T cell response to GAD, a panel of human GAD65-specific T lymphocyte lines was generated from peripheral blood of three recent onset IDDM patients. All lines derived from a patient expressing the high-risk-conferring HLA-DR*0301/ *0401 haplotypes recognized a single epitope localized between amino acid positions 270 and 283 of GAD65, a stretch that is located in close proximity to the homology region shared with Coxsackie virus P2-C protein. All lines with this specificity were restricted to the DRA, B1*0401 product of the DR4 haplotype. Analysis of the GAD-specific T cell response in a second patient homozygous for DR4 haplotypes demonstrated that the same DRA, B1*0401 allele selected T cells specific for a different determinant. The T cell response profile in a third patient showed that DR*1501/ *1601-encoding haplotypes could present at least three different epitopes to GAD65-specific T lymphocytes. One of these epitopes was presented by a DR allele associated with the resistance-conferring DRB1*1501 haplotype. GAD-specific T cell lines could not be isolated from HLA class II-matched normal individuals. Our data reveal that (a) the T cell response to GAD65 is quite heterogenous in recent onset IDDM patients; (b) HLA-DR, not DQ, seems to be the principal restriction element used by T cells present at the onset of the disease; and (c) T cells responding to epitopes containing identical sequences to Coxsackie virus P2-C protein were not detected.

Altered immune response to insulin in newly diagnosed compared to insulin-treated diabetic patients and healthy control subjects.

Insulin-dependent diabetes mellitus (IDDM) is the result of a T-cell mediated autoimmune beta-cell destruction, which is accompanied by autoantibodies. We analysed the cellular and humoral immune response to insulin and insulin peptides in patients with recent-onset IDDM, IDDM patients treated with insulin, non-diabetic first degree relatives and unrelated control subjects. There were no differences in T-cell reactivity to whole insulin or insulin peptides in general between age-matched groups of IDDM patients, relatives or healthy control subjects. In contrast to investigations in NOD mice, no immunodominant or disease-specific insulin peptide could be identified. Surprisingly, a positive correlation of T-cell responses to insulin with age was noticed (p < 0.005). This resulted in an inverse relation of insulin autoantibodies (IAA) and insulin reactive T-cells (p < 0.001) together with the well-described negative correlation of IAA with age. Interestingly, insulin-treated patients differed from age-matched recent-onset IDDM patients: first, simultaneous immune recognition of insulin with T-cells and IAA was only seen in patients treated for 6 months with insulin; second, insulin-treated patients rarely responded to whole insulin; third, they displayed less determinant spreading, and finally, recognition of multiple insulin peptides was not accompanied by crossreactivity to whole insulin. These distinct observations in insulin-treated IDDM patients, together with the inverse correlation between humoral and cellular responses to insulin, may result from activation or modulation of different T-cell subsets, and may be of relevance to insulin therapy trials, in which selective activation of non-destructive T-cell subsets may be a key to successful intervention.

T-cell reactivity to GAD65 peptide sequences shared with coxsackie virus protein in recent-onset IDDM, post-onset IDDM patients and control subjects

GAD65 is one of the major autoantigens associated with insulin-dependent diabetes mellitus (IDDM). The two peptides p17 and p18 of GAD65 that share sequence similarity with coxsackie virus (amino acid sequence identity: PEVKEK) appeared to be the major determinants of GAD65 recognized preferably by T cells from new-onset IDDM patients and their first degree relatives. In contrast, in our study unrelated control subjects frequently recognized the two GAD peptides (55%, 16/29), similar to first degree relatives (41%, 12/29) and IDDM patients post-onset (68%, 15/22). However, recent-onset IDDM patients, responded less frequently (25%, 4/16) compared with IDDM patients post-onset (p < 0.03) or unrelated control subjects (borderline significant) confirming previous observations in humans and NOD mice that T-cell reactivity to GADp17/p18 at diabetes onset is decreased. Moreover, this study demonstrated a positive correlation of T-cell proliferation to GAD p17 (amino acid 247-266) and p18 (amino acid 260-279) with simultaneous responses to both peptides in 13% of all subjects tested (n = 97) (p < 0.001). T-cell proliferation to GAD p17 was higher than to p18 in recent-onset diabetic patients, first degree relatives and unrelated control subjects (p < 0.02, p < 0.004, p < 0.002, respectively). However, in post-onset IDDM patients, the two peptides were recognized equally well. Our results show that T-cell reactivity to GAD65, peptides homologous with coxsackie protein is very frequently observed, but not primarily associated with IDDM. The temporary decline of T-cell proliferation is not associated with the beta-cell destruction process, but with clinical manifestation. The positive correlation of reactivity to the two peptides in the viral motif implicates that PEVKEK is an immunogenic epitope.

Antibodies to Bovine Serum Albumin in Japanese IDDM Children; No Association with GAD Antibodies and HLA Risk Genotypes

The implications of Bovine serum albumin (BSA) in cow's milk proteins in the pathogenesis of insulin-dependent diabetes mellitus (IDDM) have been suggested, but is controversial even in Caucasians, and have been addressed in limited studies in other ethnic groups. We studied the prevalence of antibodies to BSA (BSAAb) in sera from Japanese children with IDDM, and compared it to those in children with other diseases including non insulin-dependent diabetes mellitus (NIDDM), autoimmune thyroid disease (AITD) and congenital hypothyroidism (cretinism) by using enzymelinked immunosorbent assay (ELISA). Significant levels of BSAAb were detected in sera from non-diabetic normal children, and higher levels in younger (1-3 years of age) than in older children (4-9 years of age). Only 2 of 20 (10%) recent-onset IDDM patients of age ≥ 4 years were assessed to have high titers of BSAAb compared to normal controls, and none of 8 sera from those of age <4 years were positive for BSAAb. No significant difference in the incidence of BSAAb was found among the various groups of children examined in both age groups. It appeared that BSAAb in IDDM patients bound to different sites on BSA from ABBOS (a 17-amino acid peptide, position 152 to 168 of BSA), based on the result that no significant blocking of BSAAb by the addition of ABBOS was detected by inhibition assay. Antibodies to glutamic acid decarboxylase (GADAb) in sera and human leukocyte antigen (HLA) -DR, -DQ genotypes of 28 recent-onset IDDM patients were examined. In addition, there was no significant difference between GADAb-positive and -negative IDDM patients in the titers of BSAAb. No association of BSAAb with HLA genotypes such as HLA-DR9-DQ9 and DR4-DQ4 was found in recent-onset IDDM patients. Collectively, BSAAb was not associated with IDDM in Japanese children.

Cloning and Characterization of Islet Cell Antigen-related Protein-tyrosine Phosphatase (PTP), a Novel Receptor-like PTP and Autoantigen in Insulin-dependent Diabetes

Cloning of the cDNA encoding a novel human protein- tyrosine phosphatase (PTP) called islet cell antigen-related PTP (IAR) predicts a receptor-like molecule with an extracellular domain of 614 amino acids containing a hydrophobic signal peptide, one potential N-glycosylation site, and an RGDS peptide which is a possible adhesive recognition sequence. The 376-amino acid intracellular region contains a single catalytic domain. Recombinant IAR polypeptide has phosphatase activity. Northern blot analysis shows tissue-specific expression of two IAR transcripts of 5.5 and 3. 7 kilobases, which are most abundant in brain and pancreas. The IAR PTP is homologous in its intracellular region to IA-2, a putative PTP that is an insulin-dependent diabetes mellitus (IDDM) autoantigen. IAR is also reactive with IDDM patient sera. IAR and IA-2 may distinguish different populations of IDDM autoantibodies since they identify overlapping but nonidentical sets of IDDM patients. Thus IAR is likely to be an islet cell antigen useful in the preclinical screening of individuals for risk of IDDM.

HLA‐associated inverse correlation between T cell and antibody responsiveness to islet autoantigen in recent‐onset insulin‐dependent diabetes mellitus

Insulin-dependent diabetes mellitus (IDDM) is a T cell-dependent immune-mediated disease. Recently, a novel islet cell antigen (ICA69) recognized by autoantibodies was described. We tested T cell responsiveness to ICA69 in peripheral blood of patients with recent onset IDDM (n = 46), patients with long-standing IDDM (n = 44), non-diabetic age-matched, islet cell autoantibody- and glutamic acid decarboxylase (GAD)65 antibody-negative first-degree relatives of IDDM patients (n = 15) and rheumatoid arthritis patients (n = 22). T cell responsiveness was significantly higher in recent onset IDDM patients, compared to IDDM patients post-disease onset, non-diabetic first degree relatives and rheumatoid arthritis patients (p < 0.001). In responding IDDM patients a significant inverse correlation between T cell and autoantibody responsiveness to ICA69 was observed (p < 0.0005). Immunogenetic evaluation revealed an association of HLA-DR3 with T cell responsiveness to ICA69 (p < 0.02) and absence of ICA69-reactive autoantibodies (p < 0.04). The increased T cell reactivity to ICA69 in the absence of antibody reactivity at onset of IDMM is associated with an HLA class II immune response gene, and therefore suggestive of a genetically controlled selective activation of T helper subsets to a specific autoantigen in humans.

Islet-infiltrating t lymphocytes in insulin-dependent diabetic patients express CD80 (B7-1) and CD86 (B7-2).

Insulin-dependent diabetes mellitus (IDDM) results mainly from T cell mediated pancreatic beta cell destruction. To fully activate antigen specific T cells, current evidence suggests that two signals are required. One signal is delivered via the antigen specific T cell receptor (TCR) when engaged by major histocompatibility complex presented antigen (MHC:Ag), the other via the T cell's CD28 when engaged by CD80/86. Recent studies have demonstrated that transgenic mice expressing CD80 on their pancreatic beta cells are susceptible to autoimmune beta cell destruction. To further explore whether CD80/86 expression plays a role in IDDM pathogenesis, we analysed pancreatic biopsy specimens from 16 recent-onset IDDM patients (13 men and 3 women; age 29.7 +/- 8.8 years) for CD80/86 expression. While no biopsy revealed any islet cell specific CD80 or CD86 expression, biopsies from six of the nine patients with insulitis revealed both CD80 and CD86 expression on the islet infiltrating cells. Triple immunofluorescent staining for CD80/86, CD3, and glucagon revealed that the CD80/86-positive cells were also CD3-positive. Of the CD3-positive cells, 19.4% expressed CD80 and 21.7% expressed CD86. CD80 and CD86-positive cells were similarly distributed throughout the inflamed islets. These data suggest that CD28 engagement with CD80/86 may play a pathogenic role in the beta cell destruction underlying IDDM.

No detectable cytomegalovirus and Epstein-Barr virus genomes in the pancreas of recent-onset IDDM patients

Viral infection is assumed to trigger or exacerbate autoimmune responses against pancreatic beta cells leading to the development of insulin-dependent diabetes mellitus (IDDM). We therefore examined by polymerase chain reaction the presence of two candidate viruses, cytomegalovirus and Epstein-Barr virus, in IDDM pancreases. Pancreas tissues were obtained by biopsy under laparoscopy from 16 recent-onset IDDM patients: age 17-53 years; disease duration 0-7 months; six had flu-like symptoms before onset. Frozen sections were made and subjected to DNA amplification. DNA samples were prepared from the frozen sections and polymerase chain reaction was performed using primers specific to cytomegalovirus, Epstein-Barr virus and control gene for HLA-DP. Cytomegalovirus- and Epstein-Barr virus-infected cells were used for positive control. Southern blot analysis could detect cytomegalovirus DNA from as few as 2 x 10(-1) cytomegalovirus-infected cells and Epstein-Barr virus DNA from two Epstein-Barr virus-infected cells. This highly sensitive analysis, however, could not detect cytomegalovirus or Epstein-Barr virus genomes in pancreases of recent-onset IDDM. A single copy human gene (HLA-DP) was amplified from all IDDM pancreases indicating that DNA amplification was performed without inhibition. We conclude that cytomegalovirus or Epstein-Barr virus genomes are unlikely to exist in pancreas biopsy specimens of recent-onset IDDM patients.

High diagnostic sensitivity of glutamate decarboxylase autoantibodies in insulin-dependent diabetes mellitus with clinical onset between age 20 and 40 years. The Belgian Diabetes Registry.

Patients with adult-onset Type 1 (insulin-dependent) diabetes mellitus (IDDM) are more difficult to identify than young patients, as their clinical onset is often less acute with a questionable state of insulin dependency. Classification may be facilitated by the detection of autoantibodies that are associated with IDDM. The prevalence of islet cell autoantibodies (ICA) and insulin autoantibodies (IAA) is, however, markedly lower in adult than in young patients. The present study assesses the usefulness of antibodies against glutamate decarboxylase (GAD), as a complementary marker. Sera from 312 recent-onset IDDM patients under age 40 and 163 age-matched controls were assayed for IAA, ICA, and antibodies against recombinant GAD65 (M(r) 65,000) or GAD67 (M(r) 67,000). IAA or ICA occurred in over 90% of patients diagnosed under age 20 but only in 65% of patients between age 20 and 40. Determination of GAD65-Ab did not increase the percent antibody positive patients under age 10, but did so at older ages: from 92-98% in the 10-19 years age group, and from 65-85% in the 20-39 years age group. The determination of GAD67-Ab did not add to the information provided by the GAD65-Ab assay. Our results indicate that, alone or in combination with ICA, the GAD65-Ab assay identified more patients with an IDDM marker in the age group 20-39 years than in the group under age 20.

T cell proliferative responses to glutamic acid decarboxylase-65 in IDDM are negatively associated with HLA DR3/4.

Based on studies in spontaneously non-obese diabetic (NOD) mice, it has been suggested that the Mr 65,000 isoform of glutamic acid decarboxylase (GAD65) is of major importance in the pathogenesis of insulin-dependent diabetes mellitus (IDDM). In humans, antibodies to GAD65 are present before and at onset of the disease and in vitro T cell reactivity to GAD has also been reported. To further characterize the T cell recognition of GAD65, we incubated peripheral blood mononuclear cells from 45 newly diagnosed IDDM patients with purified recombinant human islet GAD65 and correlated the proliferative response with HLA DR haplotype and the presence of GAD65 autoantibodies. Fifty healthy individuals were studied as controls. Of the patients, 49% showed proliferative responses to GAD65 in contrast to only 4% of the controls. T cell proliferation to GAD65 was significantly more frequent in patients not being HLA DR3/4 heterozygous (19/29, 66%) as compared to HLA DR3/4 heterozygous patients (3/16, 19%) (p < 0.01). The difference was most pronounced in females with 64% (9/14) of the HLA non-DR3/4 patients being positive compared to none (0/6) of the HLA DR3/4 patients (p < 0.05). The overall frequency of GAD65 autoantibodies was 71% (32/45) with a similar distribution between patients with HLA DR3/4 (10/16, 63%) and HLA non-DR 3/4 (22/29, 76%). There was no correlation between levels of the T and B cell responses to GAD65 (r = 0.24). In conclusion, we find a proliferative T cell response to GAD65 in approximately 50% of recent onset IDDM patients and unexpectedly find the majority of responders to be HLA non-DR 3/4 heterozygous patients. No difference was observed in B cell responsiveness between the two HLA groups.

Soluble forms of intercellular adhesion molecule-1 in insulin-dependent diabetes mellitus

Summary Soluble adhesion molecules are detectable at low levels in healthy people but are increased in various disorders. However, their physiological role is unknown. Circulating intercellular adhesion molecule-1 (cICAM-1) may modulate inflammation or arise as a consequence of inflammation. We have described elevated concentrations of cICAM-1 in subjects at risk of developing insulin-dependent diabetes mellitus (IDDM), compared with recent-onset IDDM patients and healthy controls. Here we tested the ability of a monomeric soluble recombinant form of ICAM-1 (rICAM-1), to prevent the proliferation of T cells to islet-cell and other antigens. We also tested the ability of two multivalent ICAM-1-immunoglobulin (ICAM-1-Ig) fusion proteins to stop proliferation of T cells in vitro. Autoreactive T-cell proliferation was suppressed by monoclonal antibodies directed against ICAM-1 or lymphocyte-function antigen-1 (LFA-1). Furthermore, 100 μmol rICAM-1 blocked T-cell proliferation in response to an islet-cell autoantigen, and multivalent ICAM-1-Ig fusion proteins were approximately 1000-fold more effective. The usual interleukin-2-induced proliferation of T cells was unaffected by ICAM or ICAM-Ig. In addition, rICAM-1 blocked primary T-cell responses from peripheral blood mononuclear cells of newly diagnosed IDDM patients in concentrations similar to elevated cICAM-1 concentrations in individuals at risk for the disease. Thus, naturally circulating ICAM-1 may downregulate inflammation in subjects at risk of developing IDDM. Ig-ICAM-1 fusion proteins may thus provide novel means to intervene in the pathogenesis of autoimmune diseases.

Detection of GAD65 Antibodies in Diabetes and Other Autoimmune Diseases Using a Simple Radioligand Assay

Autoantibodies to glutamic acid decarboxylase (GAD) are frequent at or before the onset of insulin-dependent diabetes mellitus (IDDM). We have developed a simple, reproducible, and quantitative immunoprecipitation radioligand assay using as antigen in vitro transcribed and translated [35S]methionine-labeled human islet GAD65. By using this assay, 77% (77 of 100) of serum samples from recent-onset IDDM patients were positive for GAD65 antibodies compared with 4% (4 of 100) of serum samples from healthy control subjects. In competition analysis with unlabeled purified recombinant human islet GAD65, binding to tracer was inhibited in 74% (74 of 100) of the GAD65-positive IDDM serum samples compared with 2% of the control samples. The levels of GAD antibodies expressed as an index value relative to a standard serum, analyzed with or without competition, were almost identical (r = 0.991). The intra- and interassay variations of a positive control serum sample were 2.9 and 7.6%, respectively (n = 4). The frequency of GAD antibodies was significantly higher with IDDM onset before the age of 30 (80%, 59 of 74) than after the age of 30 (48%, 10 of 21) (P < 0.01). The prevalence of islet cell antibodies showed a similar pattern relative to age at onset. Because simultaneous occurrences of multiple autoimmune phenomena are common, we analyzed sera from patients with other autoimmune diseases. The frequency of GAD antibodies in sera positive for DNA autoantibodies (8% [2 of 25] and 4% [1 of 25] in competition analysis) or rheuma factor autoantibodies [12% (4 of 35) and 3% (1 of 35) in competition analysis] was not different from that in control samples. In contrast, in sera positive for ribonucleoprotein antibodies the frequency of GAD antibodies was significantly increased (73% [51 of 70] and 10% [7 of 70] in competition analysis [P < 0.025]). In conclusion, even large numbers of serum samples can now be tested for GAD65 antibodies in a relatively short time, allowing screening of individuals without a family history of IDDM for the presence of this marker.

Lack of Predictive Value of Islet Cell Antibodies, Insulin Antibodies, and HLA-DR Phenotype for Remission in Cyclosporin-Treated IDDM Patients

The effect of immunosuppression on the humoral immune response to islet autoantigens and exogenously administered insulin and the predictive value of islet cell cytoplasmic antibodies (ICAs), insulin antibodies (IAs), and HLA-DR phenotype for remission during immunosuppression were studied in a prospective randomized double-blind trial of cyclosporin administration in 98 newly diagnosed insulin-dependent diabetes mellitus (IDDM) patients. HLA-DR phenotype and glycosylated hemoglobin were determined at study entry, and insulin requirement, glucagon-stimulated C-peptide, ICAs, and IAs were measured at entry and after 1, 3, 6, 9, and 12 mo of follow-up. Cyclosporin therapy caused significant suppression of the prevalence and serum concentrations of ICAs and IAs. Cyclosporin-treated IDDM patients ICA+ at study entry had higher levels of stimulated C-peptide after 1 mo of study, but the increased beta-cell function was not associated with a higher frequency of insulin-free remission at 1 mo. ICA and IA status at entry did not predict cyclosporin-insulin-free remission as assessed by the prevalence of insulin-free remission or beta-cell function at 3-12 mo of study, and significant decrements in the titers or total disappearance of ICAs were not associated with an increased prevalence or duration of non-insulin-requiring remission or higher stimulated C-peptide values. There was no correlation between the serum levels of ICAs and IAs at entry and beta-cell function at 12 mo of follow-up.(ABSTRACT TRUNCATED AT 250 WORDS)

Impairment of lymphocyte-suppressive system in recent-onset insulin-dependent diabetes mellitus. Correlation with metabolic control.

Impairment of suppressor-cell activity may be important in the pathogenesis and maintenance of insulin-dependent diabetes mellitus (IDDM). In 23 recent-onset IDDM patients, lymphocyte sensitivity in vitro to theophylline was tested both in basal conditions and after improvement of metabolic control. This pharmacologic agent is mainly effective on a lymphocytic subpopulation with phenotypic and functional suppressive features. Peripheral blood lymphocytes from IDDM patients showed a loss of theophylline sensitivity, identified as inhibition of both E-rosette formation and blastogenic response to polyclonal mitogens concanavalin A (ConA) and phytohemagglutinin (PHA). An inverse relationship was demonstrated between the theophylline-induced suppression of ConA blastogenic response and blood glucose and glycosylated hemoglobin levels (P less than .01). Metabolic control seemed to be important even in relation to lymphocyte subpopulation distribution. In IDDM patients we found a significant (P less than .05) reduction of OKT4+ lymphocytes that is correlated with blood glucose and glycosylated hemoglobin levels (P less than .01). The improvement of metabolic control led to recovery of theophylline sensitivity. We suggest a deficiency in a suppressive system that could be involved in IDDM onset and the possible role of metabolic control in the impairment of some immunologic functions reported with this pathologic condition.