Histological validation of a type 1 diabetes clinical diagnostic model for classification of diabetes.

AIMS: Misclassification of diabetes is common due to an overlap in the clinical features of type 1 and type 2 diabetes. Combined diagnostic models incorporating clinical and biomarker information have recently been developed that can aid classification, but they have not been validated using pancreatic pathology. We evaluated a clinical diagnostic model against histologically defined type 1 diabetes. METHODS: We classified cases from the Network for Pancreatic Organ donors with Diabetes (nPOD) biobank as type 1 (n = 111) or non-type 1 (n = 42) diabetes using histopathology. Type 1 diabetes was defined by lobular loss of insulin-containing islets along with multiple insulin-deficient islets. We assessed the discriminative performance of previously described type 1 diabetes diagnostic models, based on clinical features (age at diagnosis, BMI) and biomarker data [autoantibodies, type 1 diabetes genetic risk score (T1D-GRS)], and singular features for identifying type 1 diabetes by the area under the curve of the receiver operator characteristic (AUC-ROC). RESULTS: Diagnostic models validated well against histologically defined type 1 diabetes. The model combining clinical features, islet autoantibodies and T1D-GRS was strongly discriminative of type 1 diabetes, and performed better than clinical features alone (AUC-ROC 0.97 vs. 0.95; P = 0.03). Histological classification of type 1 diabetes was concordant with serum C-peptide [median < 17 pmol/l (limit of detection) vs. 1037 pmol/l in non-type 1 diabetes; P < 0.0001]. CONCLUSIONS: Our study provides robust histological evidence that a clinical diagnostic model, combining clinical features and biomarkers, could improve diabetes classification. Our study also provides reassurance that a C-peptide-based definition of type 1 diabetes is an appropriate surrogate outcome that can be used in large clinical studies where histological definition is impossible. Parts of this study were presented in abstract form at the Network for Pancreatic Organ Donors Conference, Florida, USA, 19-22 February 2019 and Diabetes UK Professional Conference, Liverpool, UK, 6-8 March 2019.

Validation of a rapid type 1 diabetes autoantibody screening assay for community-based screening of organ donors to identify subjects at increased risk for the disease.

The Network for Pancreatic Organ donors with Diabetes (nPOD) programme was developed in response to an unmet research need for human pancreatic tissue obtained from individuals with type 1 diabetes mellitus and people at increased risk [i.e. autoantibody (AAb)-positive] for the disease. This necessitated the establishment of a type 1 diabetes-specific AAb screening platform for organ procurement organizations (OPOs). Assay protocols for commercially available enzyme-linked immunosorbent assays (elisas) determining AAb against glutamic acid decarboxylase (GADA), insulinoma-associated protein-2 (IA-2A) and zinc transporter-8 (ZnT8A) were modified to identify AAb-positive donors within strict time requirements associated with organ donation programmes. These rapid elisas were evaluated by the international islet AAb standardization programme (IASP) and used by OPO laboratories as an adjunct to routine serological tests evaluating donors for organ transplantation. The rapid elisas performed well in three IASPs (2011, 2013, 2015) with 98-100% specificity for all three assays, including sensitivities of 64-82% (GADA), 60-64% (IA-2A) and 62-68% (ZnT8A). Since 2009, nPOD has screened 4442 organ donors by rapid elisa; 250 (5·6%) were identified as positive for one AAb and 14 (0.3%) for multiple AAb with 20 of these cases received by nPOD for follow-up studies (14 GADA+, two IA-2A(+) , four multiple AAb-positive). Rapid screening for type 1 diabetes-associated AAb in organ donors is feasible, allowing for identification of non-diabetic, high-risk individuals and procurement of valuable tissues for natural history studies of this disease.

Increased cardiovascular risk in South African patients with Addison's disease.

Patients with Addison's disease (AD) are believed to be at risk for cardiovascular disease (CVD). South Africa, like the rest of the developing world is experiencing an increase in CVD and patients with AD may be at double the risk of their peers. We wished to explore AD patients' CVD risk factors. A cross-sectional nationwide study in South Africa of patients with AD was conducted. A cohort of 147 patients with AD and 147 healthy control subjects were matched by age, gender, ethnicity, and BMI as far as was possible. Lipoproteins and highly-sensitive C-reactive-protein (hs-CRP) were the main outcome measures. AD patients had significantly higher triglycerides; (p=0.001), lower HDLC (p<0.001), higher hs-CRP (p<0.001), and more small dense LDL; (p=0.002) than controls. Nonesterified fatty acids were lower in patients (p<0.001). Approximately 65% [95% confidence interval (CI 55.6-72.4%)] had hypercholesterolaemia, 75% (CI 64.8-81.2%) had low HDLC, and 75% (CI 68.0-84.1%) had a higher LDLC. Thirteen percent of AD patients had diabetes mellitus, but none of the risk factors differed from the nondiabetics. Only HDLC correlated positively with daily hydrocortisone dose (r=0.32; p=0.005). In conclusion dyslipidaemia is common in South African AD patients; CVD risk assessment and intervention are probably warranted in the management of these patients.

Salivary cortisol day curves in Addison's disease in patients on hydrocortisone replacement.

Using salivary cortisol (SC) measurements, cortisol exposure in Addison's disease patients on hydrocortisone replacement was determined and compared with healthy controls. Cortisol pharmacokinetics was assessed in 31 patients with Addison's disease on replacement hydrocortisone doses (median daily dose 20 mg; range 5-50 mg) and 30 healthy control subjects. Saliva samples (n=16) were collected between 08:00 and 00:00 h in 1 day, using a passive drool technique. Cortisol exposure was evaluated by noncompartmental approach. In the patients, cortisol exposure was significantly higher than in controls: median inter-quartile range (IQR) peak cortisol (C(max)) 174.5 (59.3-837.0) vs. 6.50 (4.7-19.3) nmol/l, p=0.0001; area under the curve (AUC) 390.1 (177.1-928.9) vs. 21.4 (14.6-28.4) minutes*nmol/l, p=0.0001, trough cortisol level (C(min)) 0.49 (0.49-0.96) vs. 0.49 (0.49-0.49) nmol/l, p=0.02, occurring at 480.0 (0.1-660.0) vs. 405.0 (180.0-570.0) min, p=0.56. First peak cortisol was 174.5 (53.0-754.7) vs. 6.27 (3.90-8.47) nmol/l, p=0.0001 and second peak cortisol 18.90 (5.22-76.9) vs. 3.12 (1.76-4.79) nmol/l, p=0.0001. The time to first peak cortisol differed between the 2 groups, 30 (30-75) vs. 0.1 (0.1-30) minutes; p=0.0001. At doses studied, hydrocortisone replacement therapy results in cortisol pharmacokinetics being markedly different from endogenous cortisol profiles in healthy control subjects. Addison's disease patients had significantly higher SC levels compared to healthy control subjects.

Investigation of glucocorticoid receptor polymorphisms in relation to metabolic parameters in Addison's disease.

BACKGROUND: Uncertainty exists whether glucocorticoid receptor (GCR) polymorphisms play a role in steroid-related side effects in Addison's disease (AD) patients on hydrocortisone. The polymorphisms Bcll and N363S appear to increase sensitivity to cortisol, while the ER22/23EK polymorphism has been associated with resistance to cortisol. METHOD: One hundred and forty seven AD patients, and gender, and ethnicity-matched controls were recruited in South Africa. Three polymorphisms in the GCR were studied, using PCR followed by restriction fragment length analysis. Associations with BMI, lipids, glucose and inflammatory markers were investigated. RESULTS: In both patients and controls, the Bcll polymorphism occurred more frequently in whites than in other ethnic groups studied but was not associated with any of the metabolic parameters tested. The ER22/23EK polymorphism was associated with an increased BMI in both patients (29.4 vs 24.7  kg/m²) and control subjects (26.3 vs 24.2  kg/m²). The ER22/23EK polymorphism was also associated with lower LDL cholesterol in control subjects (3.46 vs 3.93  mmol/l) and in patients (3.52 vs 4.10  mmol/l). N363S was associated with increased BMI in controls 29.9  kg/m² vs wild type 24.8  kg/m². Median hydrocortisone doses were greater in patients heterozygous for either ER22/23EK 30.0  mg or N363S 25.0  mg polymorphisms than in wild type patients 20.0  mg (both comparisons). CONCLUSION: Alterations in lipids, BMI and hydrocortisone dose were associated with two polymorphisms. Further larger studies are warranted to corroborate these findings.

Blood lymphocyte chimerism associated with IVF and monochorionic dizygous twinning: case report.

We report on dizygotic (DZ) twins, conceived by IVF and ICSI with assisted hatching, who each had a mixture of 46,XX and 46,XY cells in blood lymphocytes. The female twin had mild genitalia abnormalities but further study revealed anatomically normal reproductive anatomy. Chromosome and fluorescence in situ hybridization studies of buccal, skin and ovarian tissue were normal, as were buccal tissue DNA studies. Fetal ultrasound and fetal membrane pathology were consistent with a monochorionic, diamniotic placenta (MCDAP). These twins thus have blood chimerism but are not chimeric in the other tissues studied. The mechanism for the chimerism could be due to either placental vascular anastamoses (after the development of the haematoblast stem cells) or due to an admixture of trophoblast cells during early blastocyst development. Such trophoblast cell admixtures would be restricted to the extraembryonic tissues so that general physical development in the fetus is normal and without somatic cell chimerism. This case in combination with others previously reported suggests that in IVF conceptions, the prevalence of blood chimerism associated with twinning, and the occurrence of DZ twinning associated with MCDAP, may be higher than previously thought.

Update on major trials for the prevention of type 1 diabetes mellitus: the American Diabetes Prevention Trial (DPT-1) and the European Nicotinamide Diabetes Intervention Trial (ENDIT).

The general population risk of developing type 1 diabetes mellitus (DM), 1/300, is magnified 15-20 fold in first-degree relatives of affected individuals. Because a combination of immunologic, metabolic, and genetic markers can be used to predict the disease, multicenter prevention trials in the US (DPT-1) and Europe (ENDIT) were initiated in relatives. In the DPT-1 over 80,000 relatives under 45 years of age will be screened for ICA and then 'staged' to assess risk. High-risk subjects (>50% over 5 yr) are randomized either to 4 days intravenous insulin infusion annually followed by b.i.d. low doses of subcutaneous ultralente insulin, or to close observation. To date (September 2000), 331/340 (97%) high-risk subjects have been enrolled with the intention of detecting a 35% decrease in disease over 5 years (80% power). 280/490 (57%) of intermediate risk subjects (25-50% over 5 yr) have been randomized to oral insulin or placebo. A 50% treatment difference is sought. Anticipated enrolment for the high-risk arm will be completed by year 2001, and by 2003 for the oral arm. The ENDIT study will prospectively address whether nicotinamide will reduce the rate of progression to DM in relatives. 40,000 first-degree relatives (5-40 yr) have been screened with 552 subjects (ICA titers > or = 20 JDF U) randomized to nicotinamide or placebo. This study is designed with 90% power to detect a 35% reduction in disease (placebo group estimated at 40% risk over 5 years). Analysis of data is expected in 2003.

First-phase insulin release during the intravenous glucose tolerance test as a risk factor for type 1 diabetes.

OBJECTIVE: To determine the relationship between first-phase (1 minute + 3 minutes) insulin production during the intravenous glucose tolerance test (IV-GTT) and risk factors for developing type 1 diabetes. STUDY DESIGN: Relatives of persons with type 1 diabetes (n = 59,600) were screened for islet cell antibodies (ICAs). Subjects who had positive screening results underwent IV-GTT (> or =2 times), repeat ICA screening, insulin autoantibody (IAA) screening twice, and an oral glucose tolerance test. RESULTS: Of the 59,600 subjects in the study, 2199 (3.69%) had positive findings on initial ICA test. IV-GTTs were performed in 1622 subjects, with children <8 years having the lowest first-phase insulin release (FPIR) and subjects 8 to 20 years of age having the highest FPIR. The FPIR was lower for subjects with a confirmed positive ICA test result or a positive IAA test result, subjects with higher titers of ICA or IAA, and subjects who had an abnormal (impaired or diabetic) oral glucose tolerance test result. CONCLUSION: FPIR in the IV-GTT correlates strongly with risk factors for development of type 1 diabetes.

Prediction and prevention of type 1 diabetes.

The incidence of type 1 diabetes continues to increase worldwide. Despite major strides in the daily care of patients with the disease, the patients' contribution to overall morbidity and mortality statistics and health care economic burden to society is disproportionately large because of the high rate of microvascular and macrovascular complications. The quest for prevention of type 1 diabetes has been made feasible by the unraveling of the immunogenetics of the disease and the identification of at-risk subjects by an enhanced understanding of the natural history of the prediabetic period. A combination of immunologic, metabolic, and genetic markers can be used to accurately predict the disease in higher-risk relatives and the general population. This has enabled initiation of worldwide trials (Diabetes Prevention Trial-Type 1, European Nicotinamide Diabetes Intervention Trial, and Trial to Prevent Diabetes in Genetically at Risk) aimed at the prevention of the disease. Various promising agents are being considered for use in different at-risk populations in the near future.

Reversal of insulin-dependent diabetes using islets generated in vitro from pancreatic stem cells.

Ductal structures of the adult pancreas contain stem cells that differentiate into islets of Langerhans. Here, we grew pancreatic ductal epithelial cells isolated from prediabetic adult non-obese diabetic mice in long-term cultures, where they were induced to produce functioning islets containing alpha, beta and delta cells. These in vitro-generated islets showed temporal changes in mRNA transcripts for islet cell-associated differentiation markers, responded in vitro to glucose challenge, and reversed insulin-dependent diabetes after being implanted into diabetic non-obese diabetic mice. The ability to control growth and differentiation of islet stem cells provides an abundant islet source for beta-cell reconstitution in type I diabetes.

Islet cell antibody-positive relatives with human leukocyte antigen DQA1*0102, DQB1*0602: identification by the Diabetes Prevention Trial-type 1.

The presence of human leukocyte antigen (HLA) haplotype DQA1*0102, DQB1*0602 is associated with protection from type 1 diabetes. The Diabetes Prevention Trial-type 1 has identified 100 islet cell antibody (ICA)-positive relatives with this protective haplotype, far exceeding the number of such subjects reported in other studies worldwide. Comparisons between ICA+ relatives with and without DQB1*0602 demonstrated no differences in gender or age; however, among racial groups, African-American ICA+ relatives were more likely to carry this haplotype than others. The ICA+ DQB1*0602 individuals were less likely to have additional risk factors for diabetes [insulin autoantibody (IAA) positive or low first phase insulin release (FPIR)] than ICA+ relatives without DQB1*0602. However, 29% of the ICA+ DQB1*0602 relatives did have IAA or low FPIR. Although half of the ICA+ DQB1*0602 relatives had a high risk second haplotype, this was not associated with the additional risk factors for diabetes. Hispanic ICA+ individuals with DQB1*0602 were more likely to be IAA positive or to have low FPIR than other racial groups. In conclusion, the presence of ICA in the relatives described here suggests that whatever the mechanism that protects DQB1*0602 individuals from diabetes, it is likely to occur after the diabetes disease process has begun. In addition, there may be different effects of DQB1*0602 between ethnic groups.

The prevention of type I diabetes mellitus.

Now that prediction of type I diabetes mellitus has markedly improved, worldwide attempts to prevent the disease are under way (e.g., DPT-1, ENDIT, and TRIGR). Subjects are being recruited and families of children or parents with diabetes should be informed about the availability of such studies and given the option to participate. The creation of a network of study sites or cooperative groups will allow for the implementation of new protocols aimed at preventing the disease. The greatest barrier to the prevention of diabetes is the lack of proven effective interventional agents. The journey toward prevention of type I diabetes mellitus has only just begun.

Patient and family reflections on the use of subcutaneous insulin to prevent diabetes: a retrospective evaluation from a pilot prevention trial.

A retrospective, semi-structured telephone interview was used to collect data from 28 of 31 families who participated in a pilot study testing subcutaneous insulin as a means to prevent or delay insulin-dependent diabetes mellitus (IDDM) onset. Interviews were conducted an average of 3 years after the initiation of the subcutaneous insulin protocol. Both the high-risk person (if > or = 8 years of age) and a family member (spouse or parent) were interviewed. Most participants reported that they were distressed to learn that they or a family member were at risk for IDDM, and families readily agreed to initiate subcutaneous insulin therapy. More children than adults reported insulin injections and blood glucose tests were "hard" or "very hard," but noncompliance was more common in adults. Of the high-risk participants interviewed, 58% of children and 100% of adults reported experiencing hypoglycemia, although episodes requiring someone else's assistance were rare, occurring in 38% of the children and 27% of the adults. Participants remained enthusiastic about trial participation; most favored screening programs to identify those at risk for IDDM, believed screening should be conducted regardless of age, believed subcutaneous insulin prevented or delayed IDDM onset, and would recommend subcutaneous insulin therapy to another high-risk individual. In addition, more than 40% of children, parents, and spouses reported that they would have benefited from access to a mental health professional at some point during the trial.

Cellular immune responses to beta casein: elevated in but not specific for individuals with Type I diabetes mellitus.

Elevated cellular immune responses against the cows' milk protein beta casein have been reported in individuals with Type I diabetes mellitus, a finding supportive of the concept that cows' milk consumption may be causative for the disease. We analysed cellular immune reactivities against beta casein in newly-diagnosed Type I diabetic patients, their immediate autoantibody negative relatives, and unrelated healthy individuals in order to further elucidate the role of anti-beta casein immunity in the pathogenesis of Type I diabetes mellitus. Peripheral blood mononuclear cells were stimulated in vitro with various concentrations of three different beta casein preparations, control antigens (tetanus toxoid, mumps extract) and a mitogen (phytohemagglutinin). The frequency and/or mean simulation index of cellular proliferation against two of the beta casein preparations at high antigen concentrations (i.e. 10 or 50 microg/ml) were significantly higher in newly-diagnosed Type I diabetic subjects compared with autoantibody negative healthy control subjects. However, reactivities against beta casein in the Type I diabetic probands and their autoantibody negative relatives, individuals with a very low-rate of disease development, were almost identical. Cellular immune reactivities to other antigens were similar between the subject groups. In addition to indicating the need for appropriately matched subject populations (e.g. human leukocyte antigen (HLA) matched relatives) when analysing cellular immune responses, these findings support our previous contention that individuals genetically prone to autoimmunity may be deficient in forming tolerance to dietary antigens. However, the significance of anti-beta casein immunity as a specific causative factor in the pathogenesis of Type I diabetes mellitus remains unclear.

The relationship between humoral and cellular immunity to IA-2 in IDDM.

Autoantibodies to the neuroendocrine protein insulinoma-associated protein 2 (IA-2), a member of the tyrosine phosphatase family, have been observed in individuals with or at increased risk for IDDM. Because this disease is thought to result from a T-cell-mediated autoimmune destruction of the insulin-producing pancreatic beta-cells, we analyzed humoral and cellular immune reactivity to this autoantigen to further define its role in the pathogenesis of IDDM. Peripheral blood mononuclear cells (PBMC) from individuals with newly diagnosed IDDM or at varying levels of risk for the disease were stimulated in vitro with the entire 42-kDa internal domain of IA-2 (amino acids 603-979), a series of control antigens (glutathionine-S-transferase, tetanus toxoid, Candida albicans, mumps, bovine serum albumin), and a mitogen (phytohemagglutinin). The frequency and mean stimulation index of PBMC proliferation against IA-2 was significantly higher in newly diagnosed IDDM subjects (14 of 33 [42%]; 3.8+/-4.5 at 10 microg/ml) and autoantibody-positive relatives at increased risk for IDDM (6 of 9 [66%]; 3.9+/-3.2) compared with autoantibody-negative relatives (1 of 15 [7%]; 1.8+/-1.0) or healthy control subjects (1 of 12 [8%]; 1.5+/-1.0). The frequencies of cellular immune reactivities to all other antigens were remarkably similar between each subject group. Sera from 58% of the newly diagnosed IDDM patients tested were IA-2 autoantibody positive. Despite investigations suggesting an inverse association between humoral and cellular immune reactivities against islet-cell-associated autoantigens, no such relationship was observed (rs=0.18, P=0.39) with respect to IA-2. These studies support the autoantigenic nature of IA-2 in IDDM and suggest the inclusion of cellular immune responses as an adjunct marker for the disease.

Extreme Th1 bias of invariant Valpha24JalphaQ T cells in type 1 diabetes.

Type 1 diabetes (insulin-dependent diabetes mellitus, IDDM) is a disease controlled by the major histocompatibility complex (MHC) which results from T-cell-mediated destruction of pancreatic beta-cells. The incomplete concordance in identical twins and the presence of autoreactive T cells and autoantibodies in individuals who do not develop diabetes suggest that other abnormalities must occur in the immune system for disease to result. We therefore investigated a series of at-risk non-progressors and type 1 diabetic patients (including five identical twin/triplet sets discordant for disease). The diabetic siblings had lower frequencies of CD4-CD8- Valpha24JalphaQ+ T cells compared with their non-diabetic sibling. All 56 Valpha24JalphaQ+ clones isolated from the diabetic twins/triplets secreted only interferon (IFN)-gamma upon stimulation; in contrast, 76 of 79 clones from the at-risk non-progressors and normals secreted both interleukin (IL)-4 and IFN-gamma. Half of the at-risk non-progressors had high serum levels of IL-4 and IFN-gamma. These results support a model for IDDM in which Thl-cell-mediated tissue damage is initially regulated by Valpha24JalphaQ+ T cells producing both cytokines; the loss of their capacity to secrete IL-4 is correlated with IDDM.

Prevention of insulin-dependent diabetes mellitus: an overview of three trials.

Genetic, immune, and metabolic testing can reveal a person's risk of developing insulin-dependent diabetes mellitus (IDDM), and three large clinical trials are planned or underway to see if interventions can prevent IDDM in persons at risk. Researchers in diabetes prevention trials are screening first- and second-degree relatives of probands with IDDM for islet-cell antibodies. In the Cow's Milk Avoidance Trial, infant siblings of probands with IDDM will be randomized to receive either a baby formula containing a non-antigenic protein hydrolyzate or a standard cow's milk-based formula. The Diabetes Prevention Trial-Type I is randomly assigning subjects at high risk (more than a 50% probability of developing IDDM) to either receive insulin injections or undergo observation alone; subjects at intermediate risk (25% to 50%) will receive either oral insulin or placebo. In the European Nicotinamide Diabetes Intervention Trial, subjects receive either nicotinamide or placebo. If any of these trials show that IDDM can be prevented, then large-scale screening of children for IDDM risk factors may prove beneficial.