Strategies for identifying and predicting islet autoantigen T-cell epitopes in insulin-dependent diabetes mellitus

T cells recognize peptide epitopes bound to major histocompatibility complex molecules. Human T-cell epitopes have diagnostic and therapeutic applications in autoimmune diseases. However, their accurate definition within an autoantigen by T-cell bioassay, usually proliferation, involves many costly peptides and a large amount of blood, We have therefore developed a strategy to predict T-cell epitopes and applied it to tyrosine phosphatase IA-2, an autoantigen in IDDM, and HLA-DR4(*0401). First, the binding of synthetic overlapping peptides encompassing IA-2 was measured directly to purified DR4. Secondly, a large amount of HLA-DR4 binding data were analysed by alignment using a genetic algorithm and were used to train an artificial neural network to predict the affinity of binding. This bioinformatic prediction method was then validated experimentally and used to predict DR4 binding peptides in IA-2. The binding set encompassed 85% of experimentally determined T-cell epitopes. Both the experimental and bioinformatic methods had high negative predictive values, 92% and 95%, indicating that this strategy of combining experimental results with computer modelling should lead to a significant reduction in the amount of blood and the number of peptides required to define T-cell epitopes in humans.

Coronary artery disease, microalbuminuria and lipid profile in patients with non-insulin dependent diabetes mellitus

PURPOSE: To determine the frequency of coronary artery disease, microalbuminuria and the relation to lipid profile disorders, blood pressure and clinical and metabolic features. METHODS: Fifty-five type 2 diabetic patients (32 females, 23 males), aged 59.9±9 years and with known diabetes duration of 11±7.3 years were studied. Coronary artery disease (CAD) was defined as a positive history of myocardial infarction, typical angina, myocardial revascularization or a positive stress testing. Microalbuminuria was defined when two out of three overnight urine samples had a urinary albumin excretion ranging 20 - 200µg/min. RESULTS: CAD was present in 24 patients (43,6%). High blood pressure (HBP) present in 32 patients (58.2%) and was more frequent in CAD group (p=0.05) HBP. Increased the risk of CAD 3.7 times (CI[1.14-12]). Microalbuminuria was present in 25 patients (45.5%) and tended to associate with higher systolic blood pressure (SBP) (p = 0.06), presence of hypertension (p = 0.06) and know diabetes duration (p = 0.08). In the stepwise multiple logistic regression the systolic blood pressure was the only variable that influenced UAE (r = 0.39, r² = 0.14, p = 0.01). The h ypertensive patients had higher cholesterol levels (p = 0.04). CONCLUSION: In our sample the frequency of microalbuminuria, hypertension, hypercholesterolemia and CHD was high. Since diabetes is an independent risk factor for cardiovascular disease, the association of others risk factors suggest the need for an intensive therapeutic intervention in primary and in secundary prevention.

Ambulatory blood pressure monitoring and microalbuminuria in normotensive subjects with insulin-dependent diabetes mellitus

OBJECTIVE: To assess the association between microalbuminuria with ambulatory blood pressure monitoring in normotensive individuals with insulin-dependent diabetes mellitus. METHODS: Thirty-seven patients underwent determination of the rate of urinary excretion of albumin through radioimmunoassay and ambulatory blood pressure monitoring. Their mean age was 26.5±6.7 years, and the mean duration of their disease was 8 (1-34) years. Microalbuminuria was defined as urinary excretion of albumin > or = 20 and <200µg/min in at least 2 out of 3 urine samples. RESULTS: Nine (24.3%) patients were microalbuminuric. Ambulatory blood pressure monitoring in the microalbuminuric patients had higher mean pressure values, mainly the systolic pressure, during sleep as compared with that in the normoalbuminuric patients (120.1±8.3 vs 110.8±7.1 mmHg; p=0.007). The pressure load was higher in the microalbuminuric individuals, mainly the systolic pressure load during wakefulness [6.3 (2.9-45.9) vs 1.6 (0-16%); p=0.001]. This was the variable that better correlated with the urinary excretion of albumin (rS=0.61; p<0.001). Systolic pressure load >50% and diastolic pressure load > 30% during sleep was associated with microalbuminuria (p=0.008). The pressure drop during sleep did not differ between the groups. CONCLUSION: Microalbuminuric normotensive insulin-dependent diabetic patients show greater mean pressure value and pressure load during ambulatory blood pressure monitoring, and these variables correlate with urinary excretion of albumin.

Metabolic origin of insulin resistance in obesity with and without type 2 (non-insulin-dependent) diabetes mellitus.

A metabolic hypothesis is presented for insulin resistance in obesity, in the presence or absence of Type 2 (non-insulin-dependent) diabetes mellitus. It is based on physiological mechanisms including a series of negative feed-back mechanisms, with the inhibition of the function of the glycogen cycle in skeletal muscle as a consequence of decreased glucose utilization resulting from increased lipid oxidation in the obese. It considers the inhibition of glycogen synthase activity together with inhibition of glucose storage and impaired glucose tolerance. The prolonged duration of increased lipid oxidation, considered as the initial cause, may lead to Type 2 diabetes. This hypothesis is compatible with others based on the inhibition of insulin receptor kinase and of glucose transporter activities.

Encapsulated, genetically engineered cells, secreting glucagon-like peptide-1 for the treatment of non-insulin-dependent diabetes mellitus.

Non-insulin-dependent, or type II, diabetes mellitus is characterized by a progressive impairment of glucose-induced insulin secretion by pancreatic beta cells and by a relative decreased sensitivity of target tissues to the action of this hormone. About one third of type II diabetic patients are treated with oral hypoglycemic agents to stimulate insulin secretion. These drugs however risk inducing hypoglycemia and, over time, lose their efficacy. An alternative treatment is the use of glucagon-like peptide-1 (GLP-1), a gut peptidic hormone with a strong insulinotropic activity. Its activity depends of the presence of normal blood glucose concentrations and therefore does not risk inducing hypoglycemia. GLP-1 can correct hyperglycemia in diabetic patients, even in those no longer responding to hypoglycemic agents. Because it is a peptide, GLP-1 must be administered by injection; this may prevent its wide therapeutic use. Here we propose to use cell lines genetically engineered to secrete a mutant form of GLP-1 which has a longer half-life in vivo but which is as potent as the wild-type peptide. The genetically engineered cells are then encapsulated in semi-permeable hollow fibers for implantation in diabetic hosts for constant, long-term, in situ delivery of the peptide. This approach may be a novel therapy for type II diabetes.

Distribution of HLA-DRB1 alleles in a mixed population with insulin-dependent diabetes mellitus from the Southeast of Brazil

HLA class II genes are strongly associated with susceptibility and resistance to insulin-dependent diabetes mellitus (IDDM). The present study reports the HLA-DRB1 genotyping of 41 IDDM patients and 99 healthy subjects from the Southeast of Brazil (Campinas region). Both groups consisted of an ethnic mixture of Caucasian, African Negro and Amerindian origin. HLA-DRB1*03 and *04 alleles were found at significantly higher frequencies among IDDM patients compared to the controls (DRB1*03: 48.8% vs 18.2%, P<0.005, RR = 4.27; DRB1*04: 43.9% vs 15.1%, P<0.008, RR = 4.37) and were associated with a susceptibility to the disease. DRB1*03/*04 heterozygosity conferred a strong IDDM risk (RR = 5.44). In contrast, the HLA-DRB1*11 allele frequency was lower among IDDM patients (7.3% vs 26.3% in controls), but the difference was not significant. These data agree with those described for other populations and allow genetic characterization of IDDM in Brazil

Risk of adverse effects of intensified treatment in insulin-dependent diabetes mellitus: a meta-analysis

While the benefits of intensified insulin treatment in insulin-dependent (Type 1) diabetes mellitus (IDDM) are well recognized, the risks have not been comprehensively characterized. We examined the risk of severe hypoglycaemia, ketoacidosis, and death in a meta-analysis of randomized controlled trials. The MEDLINE database, reference lists, and specialist journals were searched electronically or by hand to identify relevant studies with at least 6 months of follow-up and the monitoring of glycaemia by glycosylated haemoglobin measurements. Logistic regression was used for calculation of combined odds ratios and 95% confidence intervals (95% CI). The influence of covariates was examined by including covariate-by-treatment interaction terms. Methodological study quality was assessed and sensitivity analyses were performed. Fourteen trials were identified. These contributed 16 comparisons with 1028 patients allocated to intensified and 1039 allocated to conventional treatment. A total of 846 patients suffered at least one episode of severe hypoglycaemia, 175 patients experienced ketoacidosis and 26 patients died. The combined odds ratio (95% CI) for hypoglycaemia was 2.99 (2.45-3.64), for ketoacidosis 1.74 (1.27-2.38) and for death from all causes 1.40 (0.65-3.01). The risk of severe hypoglycaemia was determined by the degree of normalization of glycaemia achieved (p=0.005 for interaction term), with the results from the Diabetes Control and Complications Trial (DCCT) in line with the other trials. Ketoacidosis risk depended on the type of intensified treatment used. Odds ratios (95% CI) were 7.20 (2.95-17.58) for exclusive use of pumps, 1.13 (0.15-8.35) for multiple daily injections and 1.28 (0.90-1.83) for trials offering a choice between the two (p = 0.004 for interaction). Mortality was significantly (p = 0.007) increased for causes potentially associated with acute complications (7 vs 0 deaths, 5 deaths attributed to ketoacidosis, and 2 sudden deaths), and non-significantly (p = 0.16) decreased for macrovascular causes (3 vs 8 deaths). We conclude that there is a substantial risk of severe adverse effects associated with intensified insulin treatment. Mortality from acute metabolic causes is increased; however, this is largely counterbalanced by a reduction in cardiovascular mortality. The excess of severe hypoglycemia in the DCCT is not exceptional. Multiple daily injection schemes may be safer than treatment with insulin pumps.

Persistent microalbuminuria in adolescents with type I (insulin-dependent) diabetes mellitus is associated to early rather than late puberty. Results of a prospective longitudinal study

Microalbuminuria is generally accepted to be highly predictive of overt diabetic nephropathy which is the leading cause of end-stage renal failure and, consequently, of death in patients with type 1 (insulin-dependent) diabetes mellitus (IDDM). Its early identification and therapy are exceedingly important. We studied prospectively the occurrence of microalbuminuria (MA) in relation to puberty and its pubertal stages in 164 children and adolescent patients (83 girls and 81 boys) with IDDM. Analysing 100 healthy subjects, normal values for albumin excretion (range: 0-10.1 micrograms/min/1.73 m2) according to sex and the different pubertal stages were defined. No significant difference between the groups were noted and, therefore, 20 micrograms/min per 1.73 m2 (3 SD above the mean) was generally defined as cutoff for MA. Of the patients with IDDM studied, 20% (20 females and 12 males) developed persistent MA (22.1-448.2 micrograms/min/1.73 m2) during the study period of 8 years. The first manifestation of persistent MA was in 69% (13 females and 9 males) during stages of early and midpuberty; and in 28% (6 females and 3 males) at a late pubertal stage or at the end of puberty. The only child who developed MA before the onset of puberty (range: 23.5-157.4 micrograms/min/1.73 m2) was found to have dystopic kidney. Therefore, all patients with IDDM should be screened for MA regardless of diabetes duration, sex and level of diabetes control beginning at the very first stage of puberty and neither earlier nor after puberty as suggested by the American Diabetes Association.

Macular edema in Hispanics with non-insulin dependent diabetes mellitus in Starr County, Texas: Prevalence, incidence and risk factors

This project is based on secondary analyses of data collected in Starr County, Texas from 1981 till 1991 to determine the prevalence, incidence and risk factors for macular edema in Hispanics with non-insulin-dependent diabetes in Starr County, Texas. Two studies were conducted. The first study examined the prevalence of macular edema in this population. Of the 310 diabetics that were included in the study 22 had macular edema. Of these 22 individuals 9 had clinically significant macular edema. Fasting blood glucose was found to be significantly associated with macular edema. For each 10 mg/dl increase in fasting blood glucose there was a 1.07 probability of an increase in the risk of having macular edema. Individuals with fasting blood glucose $\ge$200 mg/dl were found to be more than three times at risk of having macular edema compared to those with fasting blood glucose $<$200 mg/dl.^ In the second study the incidence and the risk factors that could cause macular edema in this Hispanic population were examined. 240 Hispanics with non-insulin-dependent diabetes mellitus and without macular edema were followed for 1223 person-years. During the follow-up period 27 individuals developed macular edema (2.21/100 person-years). High fasting blood glucose and glycosylated hemoglobin were found to be strong and independent risk factors for macular edema. Participants taking insulin were 3.9 times more at risk of developing macular edema compared to those not taking insulin. Systolic blood pressure was significantly related to macular edema, where each 10 mmHg increase in systolic blood pressure was associated with a 1.3 increase in the risk of macular edema.^ In summary, this study suggests that hyperglycemia is the main underlying factor for retinal pathological changes in this diabetic population, and that macular edema probably is not the result of sudden change in the blood glucose level. It also determined that changes in blood pressure, particularly systolic blood pressure, could trigger the development of macular edema.^ Based on the prevalence reported in this study, it is estimated that 35,500 Hispanic diabetics in the US have macular edema. This imposes a major public health challenge particularly in areas with high concentration of Mexican Americans. It also highlights the importance of public health measures directed to Mexican Americans such as health education, improved access to medical care, and periodic and careful ophthalmologic examination by ophthalmologists knowledgeable and experienced in the management of diabetic macular edema. ^

DIABETIC RETINOPATHY IN NON-INSULIN-DEPENDENT DIABETES MELLITUS IN BLACK WOMEN: A DESCRIPTIVE STUDY OF RISK FACTORS

This cross-sectional study examines the prevalence of selected potential risk factors by stage of diabetic retinopathy (DR) among Black American women with non-insulin-dependent diabetes mellitus (NIDDM) followed at a university diabetes clinic. DR was assessed by ophthalmoscopy and five-field retinography, and graded on counts of microaneurysms, hemorrhages and/or exudates, and presence of proliferative DR. Prevalence of other vascular diseases was assessed from medical records. Potential risk factors included age, known duration of diabetes, type of hypoglycemic treatment, concentrations of random capillary blood glucose, glycosylated hemoglobin, urine protein and fibrinogen, body mass index, and blood pressure. Prevalence of these risk factors is reported for three categories: No DR, mild background DR, severe background or proliferative DR (including surgically treated DR). Duration, age at diagnosis and treatment of diabetes, concentration of urine protein and average blood glucose, hypertension and cardiovascular disease were significantly associated with DR in univariate analysis. The covariance analysis employed stratification on duration, age at diagnosis and therapy of diabetes. The highest DR scores were calculated for those diagnosed before age 45, regardless of duration, therapy, or average blood glucose. Only individuals diagnosed before age 45 had high blood glucose concentrations in all categories of duration. These findings suggest that in this clinic population of Black women, those diagnosed with NIDDm before age 45 who eventually required insulin treatment were at the greatest risk of developing DR and that longterm poor glucose control is a contributing factor. These results suggest that greater emphasis be placed on this subgroup in allocating the limited resources available to improve the quality of glucose regulation, particularly through measures affecting compliance behavior.^ Findings concerning the association of DR with concentration of blood glucose and urine protein, blood pressure/hypertension and weight were compared with those reported from American Indian and Mexican American populations of the Southwestern United States where prevalence of NIDDM, hypertension and obesity is also high. Additional comparative analyses are outlined to substantiate the preliminary finding that there are systematic differences between these ethnic populations. ^

Risk factors for coronary artery disease in non-insulin dependent diabetes mellitus: United Kingdom prospective diabetes study (UKPDS: 23)

Objective: To evaluate baseline risk factors for coronary artery disease in patients with type 2 diabetes mellitus.