Heike Weiss

The LEW.1AR1/Ztm-iddm rat: a new model of spontaneous insulin-dependent diabetes mellitus.

Abstract.Aims/hypothesis: We describe a new Type I (insulin-dependent) diabetes mellitus rat model (LEW.1AR1/Ztm-iddm) which arose through a spontaneous mutation in a congenic Lewis rat strain with a defined MHC haplotype (RT1.Aa B/Du Cu).Methods: The development of diabetes was characterised using biochemical, immunological and morphological methods. Results: Diabetes appeared in the rats with an incidence of 20 % without major sex preference at 58 ± 2 days. The disease was characterised by hyperglycaemia, glycosuria, ketonuria and polyuria. In peripheral blood, the proportion of T lymphocytes was in the normal range expressing the RT6.1 differentiation antigen. Islets were heavily infiltrated with B and T lymphocytes, macrophages and NK cells with beta cells rapidly destroyed through apoptosis in areas of insulitis. Conclusion/interpretation: This Type I diabetic rat develops a spontaneous insulin-dependent autoimmune diabetes through beta cell apoptosis. It could prove to be a valuable new animal model for clarifying the mechanisms involved in the development of autoimmune diabetes. [Diabetologia (2001) 44: 1189–1196]

Prevention of spontaneous immune-mediated diabetes development in the LEW.1AR1-iddm rat by selective CD8 + T cell transfer is associated with a cytokine shift in the pancreas-draining lymph nodes

Aims/hypothesisThe LEW.1AR1-iddm rat is an animal model of spontaneous type 1 diabetes mellitus. This study analysed how adoptive transfer of selective T cell subpopulations affects the incidence of diabetes.MethodsCD4+ or CD8+ T cells were isolated from diabetic LEW.1AR1-iddm rats or diabetes-resistant LEW.1AR1 rats. Cells were selectively transferred into athymic LEW.1AR1-Whnrnu or prediabetic LEW.1AR1-iddm rats. The animals were monitored for blood glucose, islet infiltration and immune cell composition of pancreas-draining lymph nodes.ResultsAfter adoptive transfer of CD4+ T cells from diabetic LEW.1AR1-iddm rats into athymic LEW.1AR1-Whnrnu rats, 50% of the recipients developed diabetes. Transfer of CD8+ T cells failed to induce diabetes. Only 10% of the athymic recipients became diabetic after co-transfer of CD4+ and CD8+ T cells. Adoptive transfer of CD8+ T cells from LEW.1AR1 or diabetic LEW.1AR1-iddm rats into prediabetic LEW.1AR1-iddm rats significantly reduced the incidence of diabetes. In protected normoglycaemic animals regulatory CD8+/CD25+ and CD4+/CD25+ T cell subpopulations that were also FOXP3-positive accumulated in the pancreas-draining lymph nodes. In this lymphatic organ, gene expression of anti-inflammatory cytokines was significantly higher than in diabetic rats.Conclusions/interpretationOur results show that adoptive transfer of CD4+ but not CD8+ T cells from diabetic LEW.1AR1-iddm rats induced diabetes development. Importantly, CD8+ T cells from diabetic LEW.1AR1-iddm rats and diabetes-resistant LEW.1AR1 rats provided protection against beta cell destruction. The accumulation of regulatory T cells in the pancreas-draining lymph nodes from protected rats indicates that transferred CD8+ T cells may have beneficial effects in the control of beta cell autoimmunity.

Genetic analysis of the LEW.1AR1-iddm rat : an animal model for spontaneous diabetes mellitus

The LEW.1AR1-iddm/Ztm rat is a new animal model of type 1 diabetes mellitus, which shows an autosomal recessive mode of inheritance for the diabetes-inducing gene. The aim of this study was to define predisposing loci of the diabetic syndrome by linkage analysis using microsatellite markers. A backcross population of 218 rats (BN × LEW.1AR1-iddm) × LEW.1AR1-iddm was analyzed using 157 polymorphic microsatellite markers covering the entire genome. Three genomic regions showed a significant linkage to the diabetic syndrome. The first susceptibility locus on rat Chromosome (RNO) 1 (LOD score 4.13) mapped to the region 1q51–55, which codes for potential candidate genes like Ins1 and Nkx2-3. The second susceptibility locus was also localized on RNO1 in the centromeric region 1p11 (LOD score 2.7) encompassing the Sod2 gene. The third quantitative trait loci (LOD score 2.97) was located on RNO20 within the major histocompatibility complex region. Comparative mapping revealed that the homologous regions in the human genome contain the IDDM loci 1, 5, 8, and 17. The identification of diabetes susceptibility regions of the genetically uniform LEW.1AR1-iddm rat strain will pave the way toward a detailed characterization of the loci conferring diabetes development as well as their functional relevance for the pathogenesis of type 1 diabetes mellitus.

Effects of polyinosinic-polycytidylic acid and adoptive transfer of immune cells in the LEW.1AR1-iddm rat and in its coisogenic LEW.1AR1 background strain

The importance of the cellular immune system for the development of T1DM in the LEW.1AR1-iddm rat was investigated by use of polyinosinic-polycytidylic acid (Poly I:C) and by adoptive transfer of concanavalin A (Con A) activated lymphocytes from diabetic LEW.1AR1-iddm rats and the coisogenic LEW.1AR1 background strain. Poly I:C treatment induced diabetes, characterized morphologically by a diffuse infiltration of the pancreas, in up to 20% of the animals of the coisogenic LEW.1AR1 background strain. It did not increase the diabetes incidence of 30% of the LEW.1AR1-iddm strain. In contrast Poly I:C treatment induced diabetes in up to 80% of the animals of the Mhc congenic LEW.1WR1 strain. Adoptive transfer of lymphocytes activated by the T-cell mitogen Con A from diabetic donors doubled the incidence of diabetes, characterized morphologically by a focal insulitis, in diabetes prone LEW.1AR1-iddm recipients. In contrast, animals of the LEW.1AR1 background strain did not develop diabetes after adoptive transfer. Moreover, adoptive transfer of Con A activated lymphocytes from LEW.1AR1 rats to LEW.1AR1-iddm rats with 30 or 60% diabetes incidence, significantly decreased the incidence of diabetes in LEW.1AR1-iddm rats with 60% diabetes incidence. The results show that autoreactive lymphocytes induce beta cell destruction in the LEW.1AR1-iddm rat, while the LEW.1AR1 background strain apparently contains regulatory potential, which is able to counteract the autoimmune response.

A Variable CD3+ T-Cell Frequency in Peripheral Blood Lymphocytes Associated with Type 1 Diabetes Mellitus Development in the LEW.1AR1-iddm Rat

Purpose The LEW.1AR1-iddm rat is an animal model of human type 1 diabetes mellitus (T1DM), which arose through a spontaneous mutation within the MHC-congenic inbred strain LEW.1AR1 (RT1r2). In contrast to the diabetes-resistant LEW.1AR1 background strain in LEW.1AR1-iddm rats a highly variable T-cell frequency could be observed in peripheral blood lymphocytes (PBLs). Methods In this study we therefore characterised the T-cell repertoire within the PBLs of the two strains by flow cytometry analysis and identified the CD3+ T-cell phenotype and its possible linkage to diabetes susceptibility. To map loci conferring susceptibility to variable CD3+ T-cell frequency, backcross strains (N2) were generated with the genetically divergent BN and PAR rats for microsatellite analysis. Results The LEW.1AR1-iddm rat strain was characterised by a higher variability of CD3+ T-cells in PBLs along with a slightly decreased mean value compared to the LEW.1AR1 background strain. The reason for this reduction was a decrease in the CD4+ T-cell count while the CD8+ T-cell proportion remained unchanged. However, both T-cell subpopulations showed a high variability. This resulted in a lower CD4+/CD8+ T-cell ratio than in LEW.1AR1 rats. Like LEW.1AR1-iddm rats all animals of the backcross populations, N2 BN and N2 PAR rats, also showed large variations of the CD3+ T-cell frequency. The phenotype of variable CD3+ T-cell frequency mapped to the telomeric region of chromosome 1 (RNO1), which is identical with the already known Iddm8 diabetes susceptibility region. The data indicate that a variable CD3+ T-cell frequency in PBLs is genetically linked to diabetes susceptibility in the LEW.1AR1-iddm rat. Conclusion The T-cell variability in PBLs could be related to the previously reported imbalance between regulatory and effector T-cell populations which results in beta-cell autoimmunity. Since similar T-cell phenotypes have also been described in human T1DM the identification of the functional role of the observed variable CD3+ T-cell frequency may help to understand the mechanisms of autoimmunity in T1DM.

Age-Related Changes in Murine Corneal Nerves

ABSTRACT Purpose: The aim of this study is to determine age-related morphological changes in the corneal subbasal nerve plexus (SNP) in two inbred mouse strains. Materials and methods: The corneal SNP was investigated by in vivo confocal laser scanning microscopy (CLSM) in 0.5-, 1-, 1.5-, and 2-year-old C57BL/6J mice and in 0.5- and 1-year-old BALB/c mice (n = 4 per age category and strain; 10 images per mouse). Fixed corneal samples from C57BL/6J mice were also analyzed after PGP9.5 staining. Nerve fiber density (NFD) was determined using the semi-automated NeuronJ program. In addition, a new custom-designed, fully automated computerized technique based on oriented multiscale matched filtering was tested to objectify and accelerate image analysis. Results: C57BL/6J mice showed low NFD (11.7 ± 0.5 mm/mm2). Aging from 0.5 to 1, 1.5, and 2 years resulted in significant reductions in subbasal NFD by 34%, 49%, and 66%, respectively. The decline in nerve fibers revealed by in vivo CLSM together with NeuronJ quantification was confirmed by ex vivo immunohistochemical analyses. Subbasal NFD in BALB/c mice (30.0 ± 1.4 mm/mm2) was 3-fold higher than in C57BL/6J mice. Aging from 0.5 to 1 year resulted in a significant 17% reduction in NFD. With the automated approach, NFD of 22.6 ± 2.9 mm/mm2 and a 45% reduction during aging was determined from the same images. Conclusions: An age-related reduction in subbasal corneal nerve fibers was observed. The differing extent of reduction in the two mouse strains may be accounted for by genetic factors. Automated NFD quantification of corneal nerve fibers in mice appears to be a useful, reliable, objective, and time-saving tool.

Dissecting Long-Term Glucose Metabolism Identifies New Susceptibility Period for Metabolic Dysfunction in Aged Mice

Metabolic disorders, like diabetes and obesity, are pathogenic outcomes of imbalance in glucose metabolism. Nutrient excess and mitochondrial imbalance are implicated in dysfunctional glucose metabolism with age. We used conplastic mouse strains with defined mitochondrial DNA (mtDNA) mutations on a common nuclear genomic background, and administered a high-fat diet up to 18 months of age. The conplastic mouse strain B6-mtFVB, with a mutation in the mt-Atp8 gene, conferred β-cell dysfunction and impaired glucose tolerance after high-fat diet. To our surprise, despite of this functional deficit, blood glucose levels adapted to perturbations with age. Blood glucose levels were particularly sensitive to perturbations at the early age of 3 to 6 months. Overall the dynamics consisted of a peak between 3–6 months followed by adaptation by 12 months of age. With the help of mathematical modeling we delineate how body weight, insulin and leptin regulate this non-linear blood glucose dynamics. The model predicted a second rise in glucose between 15 and 21 months, which could be experimentally confirmed as a secondary peak. We therefore hypothesize that these two peaks correspond to two sensitive periods of life, where perturbations to the basal metabolism can mark the system for vulnerability to pathologies at later age. Further mathematical modeling may perspectively allow the design of targeted periods for therapeutic interventions and could predict effects on weight loss and insulin levels under conditions of pre-diabetic obesity.

Application of classification algorithms on IDDM rat data

In our study, we intend to investigate the mechanism of tolerance induction by the modulatory anti CD4 monoclonal antibody RIB 5/2 in insulin dependent diabetes mellitus rats. The aim of this investigation is to identify the key mechanisms of immune tolerance on the level of T cell, cytokine, and chemokine biomarkers in the blood, lymphatic organs, and pancreas. Additionally, it should be possible to define good biomarkers of autoimmunity and tolerance for prediction of diabetes onset. We mainly applied decision trees and later on some other classification algorithms on a rather small data set. Unfortunately, the results are not significant but are good enough to satisfy our biological partners.

Analysis of IDDM Rats

The aim of our study is to investigate the mechanism of tolerance induction by the modulatory anti CD4 monoclonal antibody RIB 5/2 in insulin dependent diabetes mellitus rats. By this approach it should be possible to identify the key mechanisms of immune tolerance on the level of T cell, cytokine, and chemokine biomarkers in blood, lymphatic organs, and pancreas; and furthermore to define biomarkers of autoimmunity and tolerance fpr prediction of diabetes onset. So far, decision trees and some other classification algorithms have been applied on data sets of twelve rats.