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.

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.

CD5+ B lymphocytes in high-risk islet cell antibody-positive and newly diagnosed IDDM subjects.

Human CD5+ B lymphocytes produce autoantibodies that bind to self- and exogenous antigens. Extremely high percentages of CD5+ B lymphocytes are present in the fetal and newborn periods, whereas they constitute only a minority of B lymphocytes in healthy adults. Increased percentages of circulating CD5+ lymphocytes have previously been demonstrated in several autoimmune diseases, including rheumatoid arthritis, progressive systemic sclerosis, Graves' disease, and Sjögren's syndrome. We measured the percentages of B lymphocytes that expressed the CD5 determinant in 93 control subjects (age range 1 day to 59 yr, mean +/- 22.6 +/- 17.7 yr), 17 subjects with newly diagnosed insulin-dependent diabetes mellitus (IDDM; range 5-29 yr, mean +/- SD 13 +/- 5.9 yr), 31 high-risk islet cell antibody (ICA)-positive nondiabetic subjects (range 4-45 yr, mean +/- SD 19.8 +/- 14.1 yr), and 13 subjects with IDDM of greater than 5 yr duration (range 10-43 yr, mean +/- SD 24.2 +/- 9.9 yr). We report that CD5+ B-lymphocyte percentages are strikingly age dependent in healthy control subjects, declining progressively from the newborn period to the middle-age years (r = -0.75, P = 0.0001). In ICA+ nondiabetic and recent-onset IDDM subjects less than 29 yr of age, the percentage of circulating CD5+ B lymphocytes fell within the 95% confidence intervals established for control subjects. However, the age-dependent rate of decline in the percentage of CD5+ B lymphocytes within the control range was slower in ICA+ and newly diagnosed IDDM subjects than in control subjects.

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.

Permanent diabetes without serological evidence of autoimmunity after transient neonatal diabetes.

OBJECTIVE: To describe a probable association between TNDM and subsequent permanent IDDM. RESEARCH DESIGN AND METHODS: A longitudinal follow-up of a single case from birth to 12 yr of age was conducted analyzing sequential OGTTs, ICAs, AIAs, anti-GAD antibodies, and other organ-specific and nonspecific antibodies. RESULTS: A small-for-gestational-age infant developed hyperglycemia at 20 h of age and required insulin therapy for the 1st 14 wk of life (TNDM). Transient hyperglycemia and ketonuria were noted again at age 2 yr 10 mo during an intercurrent illness, but OGTT was normal; and ICA, AIA, anti-GAD65 and anti-GAD67 antibodies, antithyroid microsomal, anti-gastric parietal cell, antiadrenal, antisteroidal, and antinuclear antibodies were negative 3 wk later. At age 9 yr, hyperglycemia returned and persisted in the setting of hypoinsulinemia; ICA, AIA, anti-GAD65 and anti-GAD67 antibodies, and other organ-specific and nonspecific antibodies were again negative. Insulin therapy was initiated and has been maintained over 3 yr of follow-up. CONCLUSIONS: Our case is the fifth reported with permanent diabetes occurring after resolution of TNDM. The etiology of permanent diabetes in this setting is unknown but, unlike classical IDDM, appears unrelated to autoimmunity in our patient. The true frequency of this association remains unknown.

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.

Age and family relationship accentuate the risk of insulin-dependent diabetes mellitus (IDDM) in relatives of patients with IDDM.

The international community of diabetologists is rapidly becoming involved in intervention trials aimed at preventing insulin-dependent diabetes in high risk relatives. Whereas age and relationship to a proband with insulin-dependent diabetes mellitus interacting with detected islet cell autoantibodies (ICA) are risk factors, their independent contribution to that risk remains unclear. In a prospective study of 6851 nondiabetic relatives of 2742 probands conducted between 1979-1993, we found age, but not relationship, to be a dramatic risk variable in ICA-positive persons as estimated by the Cox regression model. The 5-yr risk of insulin-dependent diabetes mellitus was 66% for those found to have ICA detectable before age 10 yr, falling progressively to less than 16% for ICA-positive relatives over age 40 yr. In ICA-negative relatives, age and relationship are independent prognostic variables.

The pathogenesis, prediction, and prevention of insulin-dependent diabetes mellitus.

The past decade has seen great advances in our understanding of the pathogenesis of IDDM. This knowledge has led to investigate use of a battery of immunologic, genetic, and metabolic tests for identifying people with prediabetes. Therapies designed to arrest autoimmunity have been associated with incomplete responses and the complications of immunosuppression. Ultimately, we hope that IDDM can be eradicated by inducing tolerance to the diabetogenic autoantigen(s).

Intervention therapies for insulin-dependent diabetes.

Treatment of insulin-dependent diabetes remains problematic since there continues to be high rates of morbidity and mortality among affected patients. Good outcomes are most likely to be more common among patients who maintain endogenous insulin reserves for the longest time following diagnosis. The disease process can now be identified in its early, pre-symptomatic stages and thus, the time has come for the investigation of preventive therapies through multicenter clinical trials. A wide variety of strategies are available and their choice should be dependent on the pathogenic stage of disease at which treatment is initiated. This stage-specific approach to prevention is discussed with a particular focus on those therapies that will soon be tested in clinical trials.

Polyclonal nature of islet cell antibodies in insulin-dependent diabetes.

Islet cell antibodies (ICA) are associated with insulin-dependent diabetes mellitus (IDD) and have been proposed as predictive markers for the disease. To determine whether ICA result from the activation of single autoreactive B-lymphocyte clones or are the result of polyclonal B-cell activation, we assayed ICA using polyvalent antisera specific to kappa or lambda light chains as well as monoclonal antibodies to IgG1, IgG2, IgG3 and IgG4 heavy chains by indirect immunofluorescence. Sera from 38 newly diagnosed IDD patients with IgG-ICA titers greater than 1:8 by end-point dilution were studied. ICA of both kappa and lambda light chains were present in all sera. The ICA were predominantly of the IgG1 subclass (38/38), although ICA were also found to be IgG2 in 53% (20/38), IgG3 in 29% (11/38) and IgG4 in 16% (6/38). The distribution of IgG heavy chains in ICA was compared to the ICA titer, age of onset of IDD and HLA-DR phenotype of the patient. No statistical correlation could be detected at a P value less than 0.05. Our findings more likely exclude the occurrence of a single aberrant lymphocyte clone secreting ICA that may have arisen by somatic mutation in individual patients. Rather, these results are consistent with the hypothesis that ICA arise by polyclonal B-lymphocyte activation as a result of a defect of immune regulation. Since human antibodies to protein antigens are found predominantly in the IgG1 subclass, our findings support the belief that the autoantigen involved in the stimulation of ICA formation is comprised, at least in part, of protein.

Scientific writing courses for pediatrics fellows.

A six-week course in scientific writing and publishing was developed for pediatrics fellows at the University of Florida College of Medicine in 1984. It covered three areas: (1) grammar, syntax, and prose style; (2) construction of scientific papers; and (3) the submissions and review process. Increasing enrollment and the requests of course graduates led to the development of a second course, Advanced Scientific Writing; both courses are now offered annually. Class materials consist of texts in scientific writing, comprehensive syllabi, and handouts; the focus is on workshop activities, exercises, collaboration with peers, and individual consultations with the instructor. At the end of each course, participants complete detailed evaluation instruments, and the data obtained are used to modify the course's structure and content the following year.

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.

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.

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.

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.

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.