Protection by dimethyl urea against hyperglycaemia,but not insulitis,in low-dose streptozotocin-induced diabetes in the mouse

Summary The protective effect of dimethyl urea, a hydroxyl radical scavenger, against low-dose streptozotocin-induced diabetes has been evaluated. Dimethyl urea was given to C57BL/KsJ mice before five daily injections of streptozotocin. The saline pre-treated control animals became gradually hyperglycaemic, whereas the dimethyl urea treated group remained normoglycaemic during the 11 week follow-up period. Two weeks after the first streptozotocin injection, six out of ten dimethyl urea-treated and 12 out of 15 saline-treated mice had insulitis. Four or 11 weeks after the streptozotocin treatment, insulitis was rare in both groups. Multiple injections of dimethyl urea only did not affect the serum glucose concentrations or the islet morphology. It is suggested that dimethyl urea protected against hyperglycaemia by reducing the -cell cytotoxic effects of the low doses of streptozotocin. An increased number of cells would thus be preserved and the animals less prone to develop diabetes, despite the presence of an inflammatory process in the pancreatic gland.     

CD8 cytotoxic T-cell clone rapidly transfers autoimmune diabetes in very young NOD and MHC class I-compatible scid mice

Summary A CD8 T-cell clone (YNK1.3) generated from acutely diabetic NOD mouse islets, showed proliferation and cytotoxicity when challenged with NOD and BALB/c islet cells and NOD-derived insulinoma cells. When 1–2 × 10⁷ YNK1.3 cells were administered to 7–10-day-old NOD mice, the cells transferred overt diabetes very rapidly in each of the 16 recipients within 4 days of cell transfer. However, of 14 recipients receiving YNK 1.3 cells above 14 days of age none became diabetic. Fluorescent dye-labelled YNK1.3 cells extensively accumulated in the islets by 36 h after transfer in 7-day-old NOD recipients, while no significant insulitis was seen in 21-day-old recipients. Over half of NOD-scid recipients (5/9) rapidly became diabetic within 5 days after transfer of 1–2 × 10⁷ YNK1.3 cells at 7 days of age, whereas only one of 12 recipients over 14 days of age became diabetic. Furthermore, YNK1.3 cells also transferred diabetes to H-2K^d-matched very young BALB/c-scid and CB17-scid mice, but not to C57BL/6-scid mice. Thus, optimally activated islet-specific CD8 T-cell clones are able to rapidly transfer diabetes to NOD and MHC class I compatible scid mice when a large enough number is administered at 7 days of age. Administration of monoclonal antibodies against adhesion molecules involved in the trafficking of lymphocytes from the circulation into the inflammatory tissues, could not prevent the cellular infiltration of YNK1.3 cells into the islets in 7-day-old NOD recipients. The results indicate that islet cells in the mouse around 7 days of age are generally susceptible to cytotoxic CD8 T cells, suggesting, therefore, that CD8 T cells may play an important role in the initiation of autoimmune diabetes in NOD mice. [Diabetologia (1997) 40: 1044–1052] Received: 18 February 1997 and in revised form: 29 April 1997     

Noninvasive mapping of pancreatic inflammation in recent-onset type-1 diabetes patients

The inability to visualize the initiation and progression of type-1 diabetes (T1D) noninvasively in humans is a major research and clinical stumbling block. We describe an advanced, exportable method for imaging the pancreatic inflammation underlying T1D, based on MRI of the clinically approved magnetic nanoparticle (MNP) ferumoxytol. The MNP-MRI approach, which reflects nanoparticle uptake by macrophages in the inflamed pancreatic lesion, has been validated extensively in mouse models of T1D and in a pilot human study. The methodological advances reported here were enabled by extensive optimization of image acquisition at 3T, as well as by the development of improved MRI registration and visualization technologies. A proof-of-principle study on patients recently diagnosed with T1D versus healthy controls yielded two major findings: First, there was a clear difference in whole-pancreas nanoparticle accumulation in patients and controls; second, the patients with T1D exhibited pronounced inter- and intrapancreatic heterogeneity in signal intensity. The ability to generate noninvasive, 3D, high-resolution maps of pancreatic inflammation in autoimmune diabetes should prove invaluable in assessing disease initiation and progression and as an indicator of response to emerging therapies.Type 1A diabetes (T1D) is a chronic autoimmune disease characterized by destruction of the insulin-producing beta cells of the pancreatic islets of Langerhans. During an initial occult phase, immunological tolerance breaks down and autoimmunity sets in, leading to leukocyte infiltration of the islets, which reduces the number and function of insulin-producing beta cells. The overt phase of clinical diabetes starts once sufficient damage has been done that insulin production is insufficient for proper glucose homeostasis. Despite advances in the identification of disease-relevant autoantigens and susceptibility genes, most clinical trials performed so far have reported no or only a modest impact on disease course (1). This lack of successful outcomes may reflect our relative ignorance of T1D pathogenesis in humans, much of our understanding of the disease having come from animal models. This situation is, to a large extent, a consequence of the difficult-to-impossible access to pancreatic tissue during and after the development of diabetes in patients.As a consequence, much effort has been devoted to identifying readily measurable biomarkers of the autoimmune state and of the progression of islet destruction. The most widely adopted indicator is autoantibodies (autoAbs) targeting islet proteins such as insulin, GAD2 (also known as “glutamic acid decarboxylase 65,” GAD65), PTPRN (“islet cell antigen 512,” IA2), and SLC30A8 (“zinc transporter-8,” ZNT8). The presence and titer of autoAbs identifies individuals at elevated risk for developing clinical disease, those with autoAbs against multiple targets being at particularly high risk (2–5). However, autoAbs are persistent, and their presence informs on an aggregate risk of progression rather than on the status of autoimmunity or islet destruction at a given time.An indicator of leukocyte infiltration of the pancreatic islets would be a more direct disease biomarker. Inflammation of human islets has been difficult to evaluate histologically, because access to material is invasive and therefore difficult. As recently reviewed (6, 7), very limited numbers of individuals have been analyzed (e.g., ref. 8), many in conditions of questionable relevance to typical pathogenesis, e.g., patients with very long-lasting T1D in whom the autoimmune attack may have waned, or individuals who died of acute ketoacidosis. In addition, the histological evaluation can be complicated by variations in sample procurement and processing. In general, insulitis seems to be more frequent in high-risk individuals or in patients presenting with T1D at a young age, but may be less extensive than in the nonobese diabetic (NOD) mouse model (likely reflecting the much faster progression of diabetes in the latter).In rodent models of T1D, insulitis is accompanied by changes in the pancreatic microvasculature (9–11). Similarly, there is evidence of increased vascular permeability as a consequence of the inflammatory process involving the islets of patients with T1D (12). The activation of mononuclear phagocytes is an early and integral part of the local inflammatory process in both cases (13, 14). We previously showed that i.v. injection of dextran-coated magnetic nanoparticles (MNPs) phagocytosed by macrophages followed by MRI could be used to visualize the pancreatic inflammatory process in the NOD mouse model and transgenic derivatives thereof (11, 15, 16). Based on these observations, we performed a small human feasibility study with a now-discontinued nanoparticle, ferumoxtran-10 (Combidex). Results from this study suggested that pancreas uptake of these nanoparticles might be a useful, noninvasive indicator of islet inflammation in human patients with T1D (17).Here, we report crucial technological advances in our approach to quantitative, noninvasive imaging of pancreatic inflammation by nanoparticle-based visualization of local macrophage accumulation. These novelties include a newer, clinically approved nanoparticle, imaging at a higher field strength, optimized pulse sequences, and improved registration and visualization tools, analogous to those used in brain mapping. These advances combine to yield a method that has considerably better resolution and discrimination and is readily exportable. We document measurably increased levels of pancreatic inflammation, as well as pronounced regional heterogeneity, in patients with recent-onset T1D.     

Effect of T-cell receptor VΒ-specific monoclonal antibodies on cyclophosphamide-induced diabetes mellitus in non-obese diabetic mice

Summary The expression of specific T-cell receptor gene segments by T lymphocytes appears to be critically important for the induction of several experimental autoimmune diseases mediated by these cells. We examined whether this situation also applied to non-obese diabetic mice by using various T-cell receptor V-specific monoclonal antibodies. No significant age- or sex-related differences were observed in V usage by peripheral and splenic T lymphocytes. CD8⁺ T lymphocytes among the islet-derived mononuclear cells isolated from 20-week-old female non-obese diabetic mice showed heterogeneity of their V gene usage. In order to examine the role of T lymphocyte subsets expressing specific T-cell receptor V gene segments in the development of diabetes mellitus, T-cell receptor V-specific monoclonal antibodies were administered to 10-week-old male non-obese diabetic mice treated with cyclophosphamide. None of the antibodies used could significantly diminish the incidence of cyclophosphamide-induced diabetes and the severity of insulitis [anti-V3 (11 of 22 mice became diabetic, 50%), anti-V5 (9 of 14, 64%), anti-V8 (9 of 21, 43%), anti-V11 (12 of 23, 52%), anti-V14 (7 of 12, 58%), and anti-V5 + anti-V11 (6 of 12, 50%)] when compared with control mice (12 of 21, 57%). In addition, there were no significant differences in T-cell receptor V usage between diabetic and non-diabetic cyclophosphamide-treated mice. These results suggest that five T-lymphocyte subsets expressing different T-cell receptor V gene segments, considered to be candidates involved in the pathogenesis of autoimmune diabetes, do not individually contribute to the development of cyclophosphamide-induced diabetes in non-obese diabetic mice.     

Immunophenotyping of insulitis in control and essential fatty acid deficient mice treated with multiple low-dose streptozotocin

Summary Multiple injections of low-dose streptozotocin induce lymphocytic insulitis and autoimmune diabetes in male CD-1 mice. Prior to the onset of insulitis, macrophages infiltrate the islets (single cell insulitis) and presumably help initiate the lymphocytic response directed at streptozotocin-induced neoantigens on islet beta cells. Essential fatty acid deficiency ameliorates the lymphocytic insulitis and prevents diabetes in this model. We hypothesize that essential fatty acid deficiency, which perturbs eicosanoid pathways and blocks the production of inflammatory mediators such as leukotriene B₄, might prevent or diminish the single cell insulitis and, thus, abrogate the lymphocytic response. The purpose of the study was to determine whether essential fatty acid deficiency causes any differences in the immunophenotype or the time course of single cell insulitis or insulitis after low-dose streptozotocin. Three to five essential fatty acid deficient and 3–5 control mice were treated with low-dose streptozotocin and killed on days 0, 3, 5, 8, 10, 12 and 15. Frozen sections of the pancreata were stained using an immunoperoxidase method with antibodies against mouse macrophages, CD4 T-lymphocytes and CD8 T-lymphocytes. Sections were assessed for the presence and severity of single cell insulitis and insulitis. Based on cell counts in the most severely involved islet from each pancreas, there was no difference in the single cell insulitis in control and essential fatty acid deficient mice. Islets from control pancreata had a higher mean grade of lymphocytic insulitis. These findings suggest that autoimmune diabetes following low-dose streptozotocin in control mice is the result of both lymphocytic and histiocytic infiltrates with subsequent beta-cell destruction. Our results do not support the hypothesis that the protective effect of essential fatty acid deficiency is due to diminished influx of macrophages into the islets. It is, however, possible that essential fatty acid deficiency deleteriously affects macrophage function and, thus, blunts the lymphocytic response. [Diabetologia (1997) 40: 1263–1268] Received: 2 June 1997 and in revised form: 11 July 1997     

The spontaneously diabetic Wistar rat (the “BB” rat)

Summary A longitudinal study of 51 weanlings from 5 litters of BB Wistar rats was undertaken to characterize the time course of the spontaneous diabetic syndrome. Nine rats developed overt diabetes. An abnormal glucose tolerance preceded the onset of the syndrome in 6 of these 9 rats. No other clinical or metabolic variable measured was predictive of the development of this syndrome. In these rats, the onset was remarkably abrupt, followed by rapid clinical deterioration with marked hyperglycaemia, glycosuria, ketonaemia and hypoinsulinaemia attained within 2 to 8 days. Pronounced insulitis was present in the early stages of the syndrome, resulting in complete B-cell destruction at the time of sacrifice at 7 to 40 days. Among the 42 littermates, 9 revealed sequential abnormalities in oral glucose tolerance tests performed at weekly intervals (to age 90–120 d) though remaining aglycosuric and maintaining normal fasting plasma glucose levels. In 7 of these rats, a milder form of the same islet inflammatory lesion seen in the overtly diabetic animals was present. Thus the new features identified are: 1) a time course of evolution from normoglycaemia to overt diabetes telescoped into a period of days, and 2) a chemical stage or form with an insulitis similar to that found in the early stages of overt diabetes.     

GAD对NOD小鼠β细胞淋巴细胞性炎症的防护作用

目的：了解GAD对NOD小鼠的1型糖尿病是否具有预防作用并探讨其免疫作用机制。方法：用猪脑GAD50μg和不完全全弗氏佐剂（FIA）50μl混合后给4周龄雌性NOD小鼠腹腔注射（ip)(n=32),同时单独注射等量FIA作为对照组（n=19),8周龄时环磷酰胺（CY）250mg/kg ip以加速糖尿病。每周测定体重、血糖20周龄处死小鼠后测定血清C－肽、观察胰腺病理和超微结构变化。结果：20周龄时，FIA对照组的糖尿病总发病率为73．58％，而GAD组仅有6．25％小鼠发生糖尿病（P＜0．01），发病时间也明显延缓，GAD组的胰岛炎症分数较FIA组明显降低，且炎症程度减轻（P＜0．05）；GAD组的C-p水平也明显高于对照组（P＜0．05）；FIA组残存胰岛的β细胞数目稀少，有核膜和内浆网扩张、核糖体脱颗粒、分泌颗粒空泡样变等超微结构异常，而GAD组胰岛β细胞数显著多于对照组（P＜0．05），且无上述超微异常。结论：独脑GAD可预防NOD小鼠胰岛炎发生，且有保护其岛β细胞超微结构的作用。     

Maternal dietary n‐6/n‐3 fatty acid ratio affects type 1 diabetes development in the offspring of non‐obese diabetic mice

Environment factors, including maternal or infant dietary nutrition have been reported to have an influence on the pathogenesis of type 1 diabetes. In the present study, to investigate the effect of maternal or post‐weaning offspring's nutrition, in particular the essential fatty acid ratio (n‐6/n‐3) on the development of type 1 diabetes, we prepared two kinds of chows with n‐6/n‐3 ratios of 3.0 (L) and 14.5 (H), and provided them to mothers of non‐obese diabetic (NOD) mice during gestation and lactation and to the offspring after weaning. The n‐6/n‐3 ratios in breast milk and erythrocyte membrane of NOD offspring became nearly the same with that of the maternal diet at 2 weeks after birth. In the L chow‐fed offspring from L chow‐fed mother (LLL), levels of insulitis were higher than those in the H chow‐fed offspring from H chow‐fed mother (HHH) at 4 weeks of age, while the levels in the LLL offspring became lower than those in the HHH after 6 weeks. Early insulin autoantibody expressions were found from 2 to 6 weeks in the HHH offspring, but not in the LLL. The LLL offspring exhibited strong suppression of overt diabetes development in regard to the onset and accumulated incidence of diabetes compared to the HHH. The study with combined L and H chows during gestation, lactation in mother and in post‐weaning offspring revealed that only the LLH chow significantly suppressed the development of diabetes with similar kinetics to LLL chow, although the other combinations may delay the onset of diabetes. The present findings suggest that n‐6/n‐3 ratio of the maternal diet during gestation and lactation rather than that of offspring after weaning strongly affects the development of overt diabetes in NOD mice.     

Preventive effect of a new immunosuppressant FK-506 on insulitis and diabetes in non-obese diabetic mice

Summary We investigated the effect of an immunosuppressant FK-506 on histological change of islets, the onset of diabetes, and the change of spleen cell subsets in female non-obese diabetic mice. Mice administered intraperitoneally with FK-506 from 5 to 20 weeks of age showed marked suppression of mononuclear cell infiltration (insulitis) at 10 weeks of age. Among the subsets of the spleen cells, a significant decrease in the population of Thyl.2-positive T cells (pan-T), L3T4-positive T cells (mainly helper/inducer), and Lyt2-positive T cells (mainly suppressor/cytotoxic) was observed in FK-506-treated mice. Furthermore, glucose tolerance of the mice at 15 weeks of age was clearly improved. Cumulative incidence observed up to 40 weeks of age was 86% in control mice and 23% in FK-506-treated mice (p<0.01). These data indicate that FK-506 has a preventive effect on insulitis and diabetes by the suppression of cell-mediated autoimmunity in non-obese diabetic mice.