Pnina Vardi

Concentration of Insulin Autoantibodies at Onset of Type I Diabetes: Inverse Log-Linear Correlation With Age

thin needle is no more difficult or painful than subcutaneous injection. Hence an intramuscular injection can be recommended (as part of an intensive educational program) to be performed when a more rapid insulin action is desired, e.g., before eating quickly absorbed carbohydrates, or in case of ketoacidotic deterioration. Depending on the thickness of the subcutaneous fat tissue layer, length of the needle, and injection technique used, the absorption of insulin in the deltoid area may follow the intramuscular route and its pharmacokinetics and biological action can thus be manipulated.

Life-Table Analysis of Progression to Diabetes of Anti-Insulin Autoantibody-Positive Relatives of Individuals With Type I Diabetes

Cytoplasmic islet cell antibody–negative (ICA−; <20 Juvenile Diabetes Foundation units, n = 1670) and ICA+ (n = 42) first-degree relatives of type I (insulindependent) diabetic individuals were studied for competitive insulin autoantibodies (ClAAs) with a radioassay. Overall, 3.7% of first-degree relatives (64 of 1712) were CIAA+. Of ICA− relatives, 2.7% (45 of 1670) exceeded the upper limit of our normal CIAA range (>39 nU/ml), and 45% (19 of 42) of ICA+ relatives exceeded this normal range. Follow-up serums for repeat CIAA determination have been obtained from 16 of the nondiabetic CIAA/ICA individuals (time between samples, 0.4–5.8 yr). Fourteen of these 16 (87%) CIAA/ICA relatives were found to still be positive on follow-up, and 2 of the relatives who were positive on the first determination were negative on their follow-up test. With a mean follow-up of ∼2 yr, 4 of 45 (9%) of the CIAA+/ICA-relatives, 5 of 23 (22%) of the ICA/CIAA-relatives, and 12 of 19 (63%) of the CIAA+/ICA+ relatives developed diabetes. Life-table analysis indicated that, overall, 53% of CIAA+ relatives become diabetic after 5 yr of follow-up versus 65% of ICA+ relatives. Also by life-table analysis, the predicted risk after 5 yr of follow-up for progression to diabetes is 17% for CIAA+/ICA− relatives, 42% for ICA+/CIAA− relatives, and 77% for CIAA/ICA+ relatives. The highest rate of progression to diabetes was found in ICA+ relatives with CIAA levels >150 nU/ml (100% projected to be diabetic within 5 yr, P < .008 vs. ICA/CIAA relatives).

The Prevalence of Cutaneous Manifestations in IDDM Patients and Their Association With Diabetes Risk Factors and Microvascular Complications

OBJECTIVE The aim of our study was to evaluate the frequency of skin manifestations, including the diabetic hand syndrome, in young IDDM patients. In addition, we studied the relation of the cutaneous manifestations to diabetes duration, glycemic control, and microvascular complications. RESEARCH DESIGN AND METHODS The frequency of skin manifestations, including the diabetic hand syndrome, were examined in 238 IDDM patients (disease duration >5 years) and 122 healthy control subjects in a cross-sectional study. In addition, we studied the relation of the cutaneous manifestations with diabetes duration, glycemic control, BMI, microvascular complications, and stratum corneum hydration using a stepwise logistic regression. RESULTS Diabetic skin manifestations were detected in 168 of 238 (71%) IDDM patients and in 18 of 122 (14%) of the control subjects. Ichthyosiform skin changes of the shins, scleroderma-like skin changes, tinea pedis, and dry scaly palms were detected in 48 vs. 7%, 39 vs. 0%, 32 vs. 7%, and 21 vs. 0.8% of the patients and control subjects, respectively. In the diabetic patients, a significant association was found between ichthyosis of the shins and sclerodermalike skin changes of the hand (P < 0.001) and between scleroderma-like skin changes and the skin dryness of the palms (P < 0.0001). When diabetic risk factors were considered, diabetes duration was significantly associated with scleroderma-like skin changes and ichthyosis of the shins (P < 0.0001). The latter was also found to be related to diabetic retinopathy (P < 0.0001). Keratosis pilaris was present in 21% of the patients versus 9% in control subjects and was found to be exclusively associated with high BMI. CONCLUSIONS Acquired ichthyosis is a common finding and the most prevalent skin manifestation in young IDDM patients. The development of several skin manifestations in insulin-dependent patients seems to be related to duration of diabetes and to development of diabetic microvascular complications.

Radioassay Determination of Insulin Autoantibodies in NOD Mice: Correlation With Increased Risk of Progression to Overt Diabetes

In an initial cross-sectional study, 29 female and 25 male nondiabetic weaned nonobese diabetic (NOD) mice of various ages (age range 30–300 days, mean 108 ± 10 days) and 11 unweaned NOD pups were evaluated for competitive insulin autoantibodies (CIAAs) with a fluid-phase radioassay. Eleven of 54 (20%) weaned NOD mice had CIAA levels above the range (>39 nU/ml) of 81 control mice. The group of NOD mice that progressed to diabetes had a significantly higher level of CIAAs than NOD mice that did not progress to diabetes (NOD mice progressing to diabetes: CIAA 63 ± 12 nU/ml; NOD mice not progressing to diabetes: CIAA 8 ± 4 nU/ml; P < .02). Seven of 11 (64%) NOD mice having CIAA concentrations exceeding the normal range progressed to diabetes, whereas only 4 of 43 (9%) NOD mice progressed to diabetes without detection of elevated CIAAs (Fishers exact test, P < .0005). The relative risk of progressing to overt diabetes with CIAA levels >39 nU/ml was therefore 17 (P < .005), giving a positive predictive value of 64%, a negative predictive value of 91%, and an overall accuracy of 85%. None of 11 unweaned NOD pups had CIAA levels above the normal range (mean −9.4 ± 4.9 nU/ml). At 6 wk of age, 37% of female NOD mice were CIAA+, whereas none of the male animals exceeded the normal range at this age (38 ± 13 vs. 5 ± 6 nU/ml, P < .05). Results of testing two samples during the follow-up of NOD mice indicated that individual NOD mice positive or negative for CIAAs generally remain positive or negative, and CIAA concentrations often increased in initially positive animals. Fourteen NOD mice were included in a prospective study, and CIAAs were evaluated at 9-wk intervals over a follow-up of 8 mo. Four of these NOD mice had elevated CIAA levels, and 3 of 4 CIAA+ mice developed overt diabetes, whereas none of the NOD mice with CIAAs <39 nU/ml developed diabetes. These results were consistent with the results of our cross-sectional study. From these data, we concluded that 1) NOD mice express CIAAs to insulin before progressing to overt diabetes, 2) these antibodies appear after weaning, and 3) CIAA analysis can identify animals with increased or decreased risk of progressing to overt diabetes.

The Novel Multifunctional, Iron-Chelating Drugs M30 and HLA20 Protect Pancreatic β-Cell Lines from Oxidative Stress Damage

Increasing evidence suggests that oxidative stress (OS)-induced pancreatic β-cell impairments is involved in diabetes and diabetic complications. Our group has recently synthesized two multifunctional nontoxic, lipophilic, iron-chelating drugs, 5-{N-methyl-N-propargylaminomethyl}-8-hydroxyquinoline (M30) and 5-{4-propargylpiperazin-1-ylmethyl}-8-hydroxyquinoline (HLA20), for the treatment of various OS-mediated pathogeneses. These compounds contain the N-propargylamine cytoprotective moiety of the antiparkinsonian drug rasagiline (Azilect) and the iron-complexing component 8-hydroxyquinoline. The aim of this research was to evaluate the protective effect of the multifunctional iron-chelating drugs on rat insulin-producing pancreatic β-cells (INS-1E and RINm) against OS-induced cytotoxicity. We found that M30 and HLA20 markedly and dose-dependently inhibited H2O2-induced cytotoxicity, associated with decreased intracellular reactive oxygen species formation and increased catalase activity. In accordance, the catalase inhibitor 3-amino-1,2,4-triazol blocked the protective action of M30 against H2O2-induced damage. Both compounds significantly increased the levels of the iron-responsive protein transferrin receptor indicating their iron-chelating effect. Further mechanistic studies showed that M30 and HLA20 attenuated H2O2-induced mitochondrial membrane potential loss, decreased the release of cytochrome c into the cytoplasm, and inhibited the activation of caspase-3, suggesting that these drugs may produce cytoprotective effects via the preservation of mitochondrial function. These results indicate that the novel drugs, M30 and HLA20 display significant cytoprotective activity against OS-induced cytotoxicity in insulin producing β-cells, which might be of therapeutic use in the treatment of diabetes mellitus.

Neonatal pig islets induce a lower T-cell response than adult pig islets in IDDM patients.

BACKGROUND Pancreatic pig islets may provide a substitute in the future for difficult to obtain human islets for transplantation in insulin-dependent diabetes millitus (IDDM) patients. However, the immune response to xenografts may significantly hamper this approach. Because neonatal tissue is believed to be less immunogenic, we examined whether the T-cell response to neonatal pig islets differs from the response to adult islets. METHODS The T-cell proliferative response to different concentrations of sonicated neonatal and adult pig islets, as well as to insulin and mitogens, was tested in 21 recent onset IDDM patients and 21 healthy controls. We determined the presence of various circulating islet autoantibodies and their association with the T-cell response in IDDM patients. RESULTS In the IDDM patients, sonicated adult pig islets (at 1 microg protein/ml) induced a significantly higher frequency (12 of 21 vs. 1 of 21, p<0.001) and magnitude (2.58+/-0.44 vs. 1.38+/-0.13, p<0.02) of positive T-cell responses than neonatal islets at the same concentration. Similar results were obtained with a 10-fold higher concentration of islet sonicate. There was no significant association between the individual T-cell responses and the presence of circulating autoantibodies in IDDM patients. CONCLUSION These results indicate that neonatal pig islets induce a lower T-cell reactivity than adult islets, suggesting that the neonatal tissue may be immunologically more suitable for future islet xenotransplantation.

Streptozotocin and Alloxan-based Selection Improves Toxin Resistance of Insulin-producing RINm Cells

The aim of our study was to develop a method for selection of subpopulations of insulin producing RINm cells with higher resistance to beta cell toxins. Cells, resistant to streptozotocin (RINmS) and alloxan (RINmA), were obtained by repeated exposure of parental RINm cells to these two toxins, while the defense capacity, was estimated by the MTT colorimetric method, and [3H]-thymidine incorporation assay. We found that RINmS and RINmA displayed higher resistance to both streptozotocin (STZ) and alloxan (AL) when compared to the parental RINm cells. In contrast, no differences in sensitivity to hydrogen peroxide were found between toxin selected and parental cells. Partial protection from the toxic effect of STZ and AL was obtained only in the parental RINm cells after preincubation of cells with the unmetabolizable 3- O-methyl-glucose. The possibility that GLUT-2 is involved in cell sensitivity to toxins was confirmed by Western blot analysis, which showed higher expression of GLUT-2 in parental RINm compared to RINmS and RINmA cells. In addition to the higher cell defense property evidenced in the selected cells, we also found higher insulin content and insulin secretion in both RINmS and RINmA cells when compared to the parental RINm cells. In conclusion, STZ and AL treatment can be used for selection of cell sub-populations with higher cell defense properties and hormone production. The different GLUT-2 expression in parental and re sistant cells suggest involvement of GLUT-2 in mechanisms of cell response to different toxins.

Islet Cell Autoantibodies: Pathobiology and Clinical Applications

Autoimmunity directed against pancreatic islet cells results in slowly progressing β-cell destruction, culminating over years in clinically manifested insulin-dependent diabetes mellitus (IDDM). Circulating serum autoantibodies directed against the endocrine cells of the islets of Langerhans arean important hallmark of this disease. Assays for these islet cell antibodies (ICA) have facilitated the investigation and understanding of several facets in the pathogenesis of autoimmune diabetes. Their applications have begunto extend into clinical practice and have opened new avenues for early preclinical prediction and preventive prophylaxsis in IDDM.

Defective activation of p21ras in peripheral blood mononuclear cells from patients with insulin dependent diabetes mellitus.

We previously reported that a decreased TCR mediated activity of the GTP-GDP binding p21ras protooncogene is associated with prediabetes in non-obese diabetic (NOD) mice. Furthermore, prevention of autoimmune diabetes is associated with reversal of the p21ras signaling defect in NOD T cells. Based on these animal studies we determined the activation of p21ras in PBMC from patients with Insulin Dependent Diabetes Mellitus (IDDM), Non-Insulin Dependent Diabetes Mellitus (NIDDM) and normal healthy controls. Stimulation by PHA induced a decrease of 3.7 +/- 1.4% and an increase of 2.44 +/- 2.3%, p < 0.02 and 2.6 +/- 1.6%,p < 0.003 in the basal unstimulated p21ras activity in the IDDM, NIDDM and normal control groups, respectively. Expression of p21ras and its regulatory elements, the GTPase activating protein p120ras-GAP and the guanine nucleotide releasing factor (GNRF) hSOS, was comparable in the three groups. The in vitro proliferative response to PHA was comparable in the IDDM and control groups: stimulation index (SI) of 8.6 +/- 2.5 and 9.4 +/- 3.5 respectively, p < 0.44. No correlations were found in the IDDM patients between the degree of p21ras activation and the mitogen induced in vitro proliferative response or the various clinical parameters including age, gender, disease duration, daily insulin requirements and metabolic control. Taken together these data indicate that PBMC from IDDM patients are characterized by a persistent impairment in the activation of their p21ras. They also suggest that p21ras stimulated activity is a sensitive and independent parameter of PBMC activation in these patients.

Selective Localization of Factor VIII Antigenicity to Islet Endothelial Cells and Expression of Class II Antigens by Normal Human Pancreatic Ductal Epithelium

We evaluated the expression of factor VIII and class II histocompatibility antigens on frozen sections of normal human and rat pancreases. The immunohistologic studies were performed with directly fluoresceinated anti-human factor VIII and monoclonal antibodies A2B5, 3G5 (anti-islet), L-243, I-2, OK1 (anti-human Ia-DR), Leu-10 (anti-human HLA-DQ), anti-human HLA-DP, and OX6 (anti-rat Ia). Islet endothelial cells of humans and Wistar, CD, and BB diabetes-prone rats could be distinguished from intra-acinar endothelial cells by markedly enhanced factor VIII immunoreactivity. Factor VIII–antibody staining of islet endothelial cells was specifically absorbed by prior incubation of anti-human factor VIII antibody with normal human plasma but not by incubation with factor VIII–deficient plasma. By double indirect immunofluorescence, normal human pancreatic ductal epithelium expressed Ia in five of six pancreases studied.