M. Hawa

Elevated serum levels of macrophage-derived cytokines precede and accompany the onset of IDDM

SummaryTo determine whether cytokines could have a role in the development of insulin-dependent diabetes mellitus (IDDM), we measured serum levels of cytokines derived from T helper 1 (interleukin-2 and interferon-γ), T helper 2 (interleukin-4 and inter-leukin-10) lymphocytes and macrophages (tumour necrosis factor-α, interleukin-1 α and interleukin-1 Β) in patients before and after the onset of IDDM. Recently diagnosed IDDM patients had significantly higher levels of interleukin-2, interferon-γ, tumour necrosis factor-α and interleukin-1 α than patients with either long-standing IDDM, non-insulin-dependent diabetes (NIDDM), Graves disease, or control subjects (p<0.05 for all). Compared with control subjects, patients with long-standing IDDM and those with NIDDM had higher interleukin-2 and tumour necrosis factor-α levels (p<0.01 for all). Interleukin-4 and interleukin-10 were detectable in sera of patients with Graves disease only, while interleukin-1 Β was not detectable in the serum of any control or test subject. To investigate whether high cytokine levels precede the onset of IDDM, we studied 28 non-diabetic identical co-twins of patients with IDDM, followed-up prospectively for up to 6 years after the diagnosis of the index. Levels of tumour necrosis factor-α and interleukin-1 α were elevated above the normal range more frequently in the eight twins who developed diabetes than in those 20 who did not (p<0.005). Analysis of T helper 1 and T helper 2 profiles of the twin groups did not reveal a clear difference between prediabetic twins and twins remaining non-diabetic. These results support the notion that T helper 1 lymphocytes may play a role in the development of IDDM. This is associated with release of macrophage-derived cytokines, which is also a feature of the prediabetic period. The lack of evidence of a dominant T helper 1 profile of cytokine release before diabetes onset suggests that additional events, activating this arm of the cellular immune response, are required in the immediate prediabetic period.

Concordance rate for type II diabetes mellitus in monozygotic twins: Actuarial analysis

Summary To determine the concordance rate for Type II (non-insulin-dependent) diabetes mellitus in monozygotic twin pairs, initially ascertained discordant for diabetes, we carried out a prospective study on 44 non-diabetic subjects, each of whom had a sibling twin with diabetes (21 men, 23 women, median age 55 years, interquartile range 47–65). The subjects were referred as discordant for Type II diabetes. The twin pairs were part of the British Diabetic Twin Study and ascertained between May 1968 and January 1998. These subjects underwent an OGTT at time of referral and periodically thereafter. The mean follow-up was 8 years (range 0–18 years) and data were collected until January 1996. The percentage of twins who developed Type II diabetes was assessed by standard actuarial life-table methods and the pairwise concordance rate, that is the proportion of concordant pairs over the sum of concordant and discordant pairs, was calculated. The observed rates of concordance for Type II diabetes at 1, 5, 10, and 15 years follow-up were 17, 33, 57, and 76 %, respectively. The concordance rate for any abnormality of glucose metabolism (either Type II diabetes or impaired glucose tolerance) at 15 years follow-up was 96 %. The concordance rate for Type II diabetes in monozygotic twins is very high even in twins initially ascertained discordant for diabetes. [Diabetologia (1999) 42: 146–150]

Immunodominant epitopes of glutamic acid decarboxylase 65 and 67 in insulin-dependent diabetes mellitus

To find the dominant epitopes of a major autoantigen in insulin-dependent diabetes mellitus (IDDM), glutamic acid decarboxylase (GAD), we studied the reactivity of peripheral blood T lymphocytes with peptides covering both major isoforms. A significant response to GAD 65 or GAD 67 peptides was detected in 13 of 15 IDDM patients and in 9 of 10 normal controls. Controls most frequently recognised the central region of GAD 65 (residues 161-243). IDDM patients preferentially recognised residues 473-555 (p < 0.03). T-cell responses to GAD 67 peptides were similar in IDDM patients and controls. T lymphocytes from IDDM patients recognise a distinct dominant epitope of GAD, which may be an important target for the disease process.

Value of Antibodies to Islet Protein Tyrosine Phosphatase–Like Molecule in Predicting Type 1 Diabetes

Islet antigens associated with type 1 diabetes include a recently identified protein tyrosine phosphatase–like molecule IA-2, which contains the intracellular fragment IA-2ic. To determine whether combinations of antibodies including those to IA-2 characterize and predict type 1 diabetes, we studied antibodies to IA-2, IA-2ic, glutamic acid decarboxylase (GAD65), and islet cell antibodies (ICAs) in 1) 60 newly diagnosed type 1 diabetic patients followed for 1 year, 2) 31 monozygotic twin pairs discordant for type 1 diabetes followed up to 12 years (11 twins developed diabetes), 3) 18 dizygotic twin pairs discordant for type 1 diabetes, and 4) normal healthy control subjects. Newly diagnosed type 1 diabetic patients frequently had antibodies to IA-2 (62%), IA-2ic (67%), GAD65 (77%), and ICAs (85%). The intracellular fragment of IA-2 probably contains the immunodominant epitope as 137 of 143 samples with IA-2 antibodies from type 1 diabetic patients also had IA-2ic antibodies. Monozygotic twins were usually discordant for antibody specificities. Concordance was higher in monozygotic than matched dizygotic twins for both antibody combinations (33 vs. 6%, P < 0.05) and the development of diabetes (33 vs. 0%, P < 0.01). In monozygotic twins, all the antibodies were highly predictive of type 1 diabetes (positive predictive values all >87%), although antibodies were also detected in twins at low risk of disease. In summary, IA-2 emerges as a major antigen associated with type 1 diabetes and distinct from GAD65. Type 1 diabetes–associated autoimmunity, which is probably induced by environmental factors, does not necessarily herald progression to the disease. However, genetic factors may influence the development of combinations of disease-associated antibodies and the progression to type 1 diabetes.

Immune reactivity to glutamic acid decarboxylase 65 in stiff-man syndrome and type 1 diabetes mellitus

BACKGROUNDnThe immune response to an isoform of glutamic acid decarboxylase (GAD), GAD65, is associated with two clinically distinct diseases, stiff-man syndrome (SMS) and type 1 (insulin-dependent) diabetes mellitus. We sought to identify differences in the cellular and humoral immune responses to GAD in these two diseases.nnnMETHODSnWe compared T-cell responses in 14 SMS patients with axial disease and 17 patients with type 1 diabetes.nnnFINDINGSnPeripheral blood T cells of eight SMS patients recognised different immunodominant epitopes of GAD65 compared with T cells from 17 patients with type 1 diabetes. GAD regions 81-171 and 313-403 induced a dominant T-cell response in six of eight patients with SMS but in only one of 17 patients with type 1 diabetes (p=0.001). No SMS patients responded dominantly to GAD fragments 161-243 and 473-555 compared with ten patients with type 1 diabetes (p=0.008). GAD antibodies were detected in 11 of 14 SMS patients (seven with diabetes) and 11 of 17 patients with type 1 diabetes; IgG1 was dominant in both groups. SMS patients, however, were more likely than patients with diabetes to have isotypes other than IgG1 (p=0.03), in particular, IgG4 or IgE isotypes, which were not detected in patients with type 1 diabetes (p=0.012).nnnINTERPRETATIONnOur findings indicate differences between patients with SMS and type 1 diabetes in cellular (epitope recognition) and humoral (isotype pattern) responses to GAD65. Thus the same autoantigen can elicit distinct immune responses in patients with SMS, even when associated with diabetes, compared with patients with type 1 diabetes.

Cytokine secretion patterns in twins discordant for Type I diabetes

Aims/hypothesis. The search for T-cell reactions that are associated with disease in Type I (insulin-dependent) diabetes mellitus is severely hampered because control groups cannot be matched for relevant immune response genes. We therefore compared T-cell responses between identical twins discordant for Type I diabetes. Methods. Pairs of monozygotic twins (n = 17) discordant for Type I diabetes were studied. Cultures were set up from whole blood immediately after sampling and cells were challenged with human recombinant hsp60, with the mitogen phytohaemagglutinin or with the staphylococcal superantigen. Supernatants were removed after 48 or 96 h and analysed for T-helper1 type cytokines interferon-γ, TNFα and T-helper2 type cytokines IL-4, IL-10 by sandwich-ELISA. Results. The height of the T-helper1 type cytokine response to hsp60, phytohaemagglutinin or staphylococcal enterotoxin B did not show disease association, i. e. it was similar between discordant twins. In contrast, the production of T-helper2 type cytokines differed between discordant twins. The IL-10 response to hsp60 was higher in twins at low disease risk (islet cell antibody-negative) than in their diabetic cotwins (p < 0.01), as was the IL-4 response to phytohaemagglutinin (p < 0.05). No difference was seen in the cytokine response between islet cell antibody-positive twins and their diabetic cotwins. Conclusions/interpretation. The data indicate an association between T-helper2 type cytokine secretion patterns and disease or disease risk. [Diabetologia (1999) 42: 1080–1085]

Principles of Autoantibodies as Disease-specific Markers

The immune system is designed by nature to protect us from our environment. But activation of the immune response not only protects us from disease, as in infectious diseases, it can cause disease, as in autoimmunity. Indeed, about 20% of North American and European populations suffer from autoimmune or inflammatory diseases mediated by this immune response. The normal immune response involves activation of both innate and adaptive immune systems. Adaptive immunity is associated with activation of both T and B cells, the latter producing antibodies. Autoantibodies can be detected in the sera and, in recent years, have been valuable in both the diagnosis and the prediction of disease. It is our opinion that the practical value of autoantibodies has not been fully realised in the majority of diseases in which they can be detected. Realising the clinical potential of autoantibodies could be extremely valuable, and in this article we set out the principles for measuring autoantibodies both as diagnostic markers of disease activity and disease classification or as predictive markers of progression to clinical disease. Observations supporting this position are presented and whilst they are broadly based on the study of autoimmune diseases, it remains possible that the general principles they encompass can be applied to other disease processes, including cancer. Studies using autoantibodies in autoimmune diseases suggest several principles. First, autoantibodies can reflect the disease process and, when detected during a prolonged disease prodrome, autoantibodies can predict clinical disease. Second, when there is aetiological heterogeneity in clinical disease, autoantibodies can help classify the disease process. Both these points are particularly relevant when autoantibodies themselves have the capacity to damage tissue. Third, a number of distinct autoantibodies are associated with any one disease, and some are more predictive of progression to clinical symptoms than others. Fourth, the risk of progression to disease, the rate of progression, and the severity of the clinical disease can be predicted to a degree by the number of autoantibodies, the type of autoantibody and the titer of the autoantibody. – 8] In addition, associated features including genetic risk and evidence of target organ failure can enhance the predictive value of autoantibodies. These points will now be set out in more detail.

Humoral and cellular autoimmune responses in stiff person syndrome.

Abstract: Stiff person syndrome (SPS) is a chronic autoimmune disease associated with humoral and cellular immune responses to glutamic acid decarboxylase (GAD) 65. Another chronic autoimmune disease, type 1 diabetes (T1D), is also associated with autoimmune responses to this antigen, but T1D patients develop SPS only extremely rarely and only a third of SPS patients develop T1D (mostly mild manifestations in adulthood). In a previous study, we described important differences between T1D and SPS in the autoimmune response to GAD 65: (1) T cells of SPS patients recognize epitopes in the middle of GAD 65 (amino residues 81–171 and 313–403), whereas patients with T1D preferentially recognize another middle (161–243) and a C‐terminal region (473–555); and (2) GAD antibodies (Abs) were nearly exclusively of the Th1‐associated IgG1 type in T1D, whereas SPS patients had both Th1‐ and Th2‐associated IgG4 and IgE GAD Abs. These differences were not simply related to different HLA alleles. Fine epitope mapping revealed further distinct T cell epitopes in both diseases despite similar HLA background. Therefore, a single autoantigen can elicit different immune responses causing distinct chronic autoimmune diseases possibly related to a Th1 or Th2 bias of the disease.

Altered islet Beta-cell function before the onset of Type 1 (insulin-dependent) diabetes mellitus

SummaryTo define the glucose to insulin dose-response relationship before the onset of diabetes, we studied 22 nondiabetic co-twins of patients with Type 1 (insulin-dependent) diabetes mellitus and nine control subjects. All had intravenous glucose tests at 0.02, 0.1 and 0.5 g/kg and were followedup prospectively for at least 6 years. Seven twins developed diabetes a mean of 7 months later; the remaining 15 are now unlikely to develop diabetes. The seven pre-diabetic twins had higher fasting insulin levels than control subjects (4.2±2.0 vs 1.8±1.8 nmol/l; p<0.05); but lower glucose clearance (1.0±0.5 vs 1.9±0.7 %/min; p<0.05), first phase insulin response at 0.5 g/kg (21.1±23.2 vs 143±50 nmol/l; p<0.0001), and total insulin responses at 0.1 g/kg (p<0.05) and 0.5 g/kg (p<0.00005). Using a curve-fitting programme, the normal glucose to insulin relationship was lost in prediabetic twins who had lower coefficient of determination (R2) than control subjects (p<0.01). In contrast, 15 low-risk twins and their nine control subjects had similar fasting glucose and insulin levels, glucose clearance, R2 and insulin secretory responses to different glucose loads. The positive predictive values of subnormal R2 and subnormal first phase insulin response were 67 % and 58 % respectively. These observations demonstrate an altered glucose to insulin dose-response relationship and loss of maximum insulin secretory response to glucose before the onset of Type 1 diabetes.

Prognostic relevance of pancreatic uptake of technetium-99m labelled human polyclonal immunoglobulins in patients with type 1 diabetes

Abstract. Insulin-dependent type 1 diabetes (IDDM) is caused by the autoimmune destruction of insulin-producing beta cells. Approximately 10%–20% of patients may benefit from adjuvant immunotherapy upon diagnosis of the disease in order to protect residual beta-cell function. It has been suggested that this subgroup of patients differs from others by virtue of the presence of residual pancreatic inflammation and beta-cell function. In this study we have investigated to what extent technetium-99m-labelled human polyclonal immunoglobulins (99mTc-HIG) accumulate in the pancreas of IDDM patients at the time of diagnosis and 1 year thereafter, with a view to ascertaining whether HIG scintigraphy is useful for the identification of IDDM patients with residual pancreatic inflammation. Patients with recent-onset IDDM (n=15) were investigated at the time of diagnosis and 1 year later, and ten age- and sex-matched normal subjects were also studied. Gamma camera imaging and target to background ratio, analysed blind by three independent readers, were used to quantify the radioactivity in the pancreatic region and findings were correlated with metabolic, immunological and clinical parameters. Seven out of 15 newly diagnosed IDDM patients showed a significant accumulation of radiolabelled HIG in the pancreas (pancreas/bone ratio higher than the mean +2SD of normal subjects). One year after diagnosis, pancreatic accumulation of HIG was still detectable in most IDDM patients who were positive at the time of diagnosis. Six out of seven patients with positive scintigraphy had a partial clinical remission. These results indicate that HIG scintigraphy at the time of onset of diabetes identifies a subset of patients with residual beta-cell function who may benefit from adjuvant immunotherapy.