Model Of Human Type Research Articles

Systemic NKT cell deficiency in NOD mice is not detected in peripheral blood: implications for human studies.

In the diabetes-prone NOD mouse, there is a proven association between a systemic deficiency of NKT cells and the onset of type 1 diabetes. Numerous reports of similar defects within the NKT cell compartment of human type 1 diabetes patients suggested NKT cell levels might be a valuable predictor of susceptibility and could provide a target for therapeutic intervention. Two recent studies, however, found no association between type 1 diabetes and blood NKT cell levels in humans and consequently rejected a link between the onset of diabetes and NKT cell deficiency. This cast considerable doubts on the potential for NKT cell-based clinical applications and challenged the validity of the NOD mouse as a model of human type 1 diabetes. We now report that NKT cell levels in blood are a poor representation of those in other organs. Strikingly, systemic NKT cell deficiencies were identified in NOD mice with normal, or even raised, blood levels. This re-establishes the correlation between NKT cell deficiency and type 1 diabetes and raises important questions regarding the assaying of NKT cell levels in humans.

Antiproliferative effect of fluvastatin and thiazolidinedione in mesangial cells of diabetic rats.

Treatment with hydroxymethylglutaryl coenzyme A reductase inhibitors and thiazolidinedione derivatives may prevent the development of diabetic nephropathy. The precise mechanisms of the beneficial effects of these agents in mesangial cells are uncertain. We cultured mesangial cells from Otsuka Long-Evans Tokushima Fatty (OLETF) rats, a model for human type 2 diabetes mellitus. The effects of fluvastatin and/or troglitazone on DNA synthesis were determined. Fluvastatin in combination with troglitazone markedly inhibited DNA synthesis and induced apoptosis in mesangial cells from OLETF rats. Combined therapy with fluvastatin and thiazolidinedione derivatives may be effective for suppression of mesangial cell proliferation in the early phase of diabetes, thereby possibly slowing the evolution of diabetic glomerulopathy.

Increase in hepatic mRNA expression of coagulant factors in type 2 diabetic model mice

Aim: The purpose of the study is to investigate whether hypercoagulation in diabetes is observed not only in increased plasma levels of the coagulative factors but also in increased hepatic mRNA levels. Materials and methods: The age-related changes of coagulation factors were determined using KK and KK-A y mice as a model for human type 2 diabetes mellitus. Expression of the α-, β- and γ-chains of fibrinogen-mRNA and prothrombin-mRNA from the mouse liver was examined by reverse transcription–polymerase chain reaction (RT-PCR). Results: Hemoglobin A 1c (HbA 1C), plasma fibrinogen, triglyceride and insulin levels increased apparently, especially in KK-A y mouse at 4 months old. The mRNA levels of γ-chain of fibrinogen significantly increased at 3 and 4 months of age in both mice. The mRNA levels of α- and β-chains of fibrinogen and prothrombin were significantly increased in 4-month-old KK-A y mouse. Conclusions: These results suggest that the increase in hepatic mRNA expression of coagulant factors contributes to the hypercoagulable state in type 2 diabetic mice.

Genetic susceptibility in type 1 diabetes and its associated autoimmune disorders.

Animal models of type 1 diabetes There are several animal models of type 1A diabetes (immune mediated). The first inbred animal model evolved from the discovery of diabetic animals in an outbred colony of Wistar rats maintained at the Bio Breeding Research Laboratories in Ottawa, Canada. The Bio Breeding (BB) rat develops spontaneous immune-mediated diabetes with at least four loci contributing to disease: the major histocompatibility complex (MHC, iddm2) on rat chromosome 20; the lymphopenic locus iddm1 on rat chromosome 4 [1], iddm3 on rat chromosome 2 [2], and a fourth locus on rat chromosome 15. The lymphopenic phenotype (iddm1 or lyp) locus has been mapped to a syntenic region of rat chromosome 4 [1,3]. Mutation at this locus in a homozygous state produces a severe T cell lymphopenia. Hornum and co-workers have mapped the gene to a 0.3-cM region between markers D4Got59 and Abp1 and constructed a 550-kb P1-derived artificial chromosome (PAC) contig covering the region [4]. They found a frameshift mutation in Immune-Associated Nucleotide4 (Ian4) gene which encodes a GTP-binding protein localized in the outer mitochondrial membrane [5]. This mutation results in a truncated gene product that lacks twothirds of the total protein. They hypothesized that Ian 4 is involved in the regulation of T-cell apoptosis, making it an excellent candidate for iddm1. The Ian4 gene product is anchored to the outer mitochondrial membrane, and this organelle is an important site for the regulation of cell death [6]. The same mutation was found by MacMurray and co-workers but they termed the gene Ian5 [7]. Some Ian genes are expressed in mature T cells and switched on during thymic T-cell development [8–10]. The Komeda diabetes-prone (KDP) rat [11], a diabetesprone substrain of the Long-Evans Tokushima lean (LETL) rat is characterized by autoimmune destruction of pancreatic β cells [12], rapid onset of overt diabetes with no sex difference, and no significant T cell lymphopenia. The gene Cblb (Casitus B-lineage lymphoma b, also known as Cas-Br-M murine ectropic retroviral transforming sequence b) was a potential candidate given its influence on immune function. Yokoi et al. [13] confirmed a C → T homozygous nonsense mutation (arginine to a stop at codon 455, Arg455X) that removes 484 amino acids, including the proline-rich region and leucine zipper domain. The mutation is specific to the KDP rat and its original LETL strain. Approximately 80% of the Iddm/kdp1 homozygous mutant animals develop diabetes with severe insulitis, and most of the remainder showed mild to moderate insulitis. Neither heterozygous nor wildtype animals developed diabetes. They demonstrated that Cblb is a important regulator of autoimmunity and Cblb is a major susceptibility gene for rat type 1 diabetes [14]. The non-obese diabetic mouse is the best studied spontaneous animal model of type 1 diabetes. Diabetes susceptibility in the NOD is polygenic, with a major influence of genes within the MHC. Linkage analyses have identified at least 19 susceptibility loci (Idd1-Idd19) that contribute to disease pathogenesis [15,16]. Lymphocytes mediate the specific destruction of insulin-producing β cells. These loci can either confer susceptibility or resistance to expression of insulin autoantibodies, insulitis and or disease development. Idd1 has been mapped to the MHC locus on mouse chromosome 17 [17,18]. Idd1 mapped to class II I-A and I-E regions as a gene complex [19], but the incidence of the disease is strongly affected by a gene or genes outside of this segment (Idd16). Variants of the beta-2 microglobulin gene is one of the first non-MHC genes to by clearly identified. Though neither the insulin 2 gene on chromosome 7 nor the insulin 1 gene on chromsome 19 show linkage to diabetes, in crosses with normal mouse strains, knockouts of either of these genes greatly influences the development of diabetes. Several new mouse models of human type 1 diabetes have been developed using transgenic mice. We found

A null mutation in the cystatin M/E gene of ichq mice causes juvenile lethality and defects in epidermal cornification.

Cystatin M/E (CST6 ), a new member of the cystatin gene family, has a restricted expression pattern in humans, which is largely limited to cutaneous epithelia. Although cystatin M/E possesses two distinct biochemical properties, being a cysteine proteinase inhibitor and a substrate for transglutaminase, its physiological function is unknown. Here we report the isolation and characterization of the mouse Cst6 orthologue and the assignment of the chromosomal localization to the proximal end of mouse chromosome 19. This region corresponds to the locus of the spontaneous harlequin ichthyosis (ichq) mouse mutation, for which no causative gene has been identified so far. We found a nonsense mutation in the Cst6 gene of BALB/cJ-ichq/+ mice, which precludes the synthesis of functional protein. Immunohistochemistry confirmed the absence of cystatin M/E at the protein level in ichq/ichq mice. Mice that are homozygous for two null alleles display a hyperplastic, hyperkeratotic epidermis and abnormal hair follicles, and die between 5 and 12 days of age. In wild-type mice, cystatin M/E was found in the stratum granulosum and in the infundibulum of the hair follicle indicating that the anatomical site in the skin where cystatin M/E is normally expressed correlates with the abnormalities at the tissue level in ichq/ichq mice. Our data provide evidence that cystatin M/E is required for viability and for correct formation of cornified layers in the epidermis and hair follicles. The ichq mouse mutation may serve as a model for human type 2 harlequin ichthyosis.

A murine interleukin-4-Ig fusion protein regulates the expression of Th1- and Th2-specific cytokines in the pancreas of NOD mice.

Interleukin (IL)-4 is a key cytokine in T-helper type 2 (Th2) immune response. We have constructed a dimeric IL-4 molecule consisting of the murine IL-4 and the murine Fc part of IgG2a. We first tested the biological activity of the chimeric protein by in vitro studies using isolated murine spleen cells. IL-4-Ig was found to downregulate LPS-induced IFN-gamma and TNF-alpha production. The immunomodulatory potential of the fusion protein was also analyzed in non-obese diabetic (NOD) mice, an animal model for human type 1 diabetes. Female NOD mice aged 10 weeks were treated once with cyclophosphamide to accelerate and synchronize the progression of insulitis. Treatment with IL-4-Ig induced strong modulation of the pancreatic cytokine balance. Expression was downregulated for both Th1-specific cytokine IFN-gamma and the Th2-specific cytokine IL-10. IL-12p40 expression was only slightly affected. Interestingly, infiltration increased in the islets of IL-4-Ig-treated animals, and therefore did not correlate with the decreased IFN-gamma expression. Hence, IL-4-Ig did not prevent the progression from peri- to intra-insulitis, but suppressed inflammatory cytokine production. In most experiments, the biological activity of IL-4-Ig and IL-4 was comparable. We conclude that treatment with the chimeric protein IL-4-Ig effectively downregulates Th1 responses but simultaneously augments intra-islet infiltration.

1034. Frequency and Time Course for the Repopulation of Hematopoietic Stem Cell-Derived Hepatocytes in a Mouse Model of Human Type I Tyrosinemia

1034. Frequency and Time Course for the Repopulation of Hematopoietic Stem Cell-Derived Hepatocytes in a Mouse Model of Human Type I Tyrosinemia

Transgenic animal models for type 1 diabetes: linking a tetracycline-inducible promoter with a virus-inducible mouse model.

Autoimmunity is thought to emerge as a consequence of genetic predispositions and environmental tiggering factors. Often the etiology and the mechanisms involved in the autoaggressive destruction of self-components are rather complex and in many cases poorly understood. Chemokines and cytokines are central mediators of inflammatory processes that are involved in initiation and progression of autoimmunity. Many animal models for human autoimmune diseases use transgenic technology to express chemokines and/or cytokines in an organ or tissue specific manner. However, most of these model systems express the transgene irreversibly without considering the time of expression as a very important parameter. Here, we review experiences that were made from using a tetracycline-inducible promotor system (tTA-system) to express TNFalpha at various times during an ongoing autoimmune process, such as the destruction of pancreatic beta-cells in a mouse model for human type 1 diabetes.

Adeno-associated virus vector-mediated IL-10 gene delivery prevents type 1 diabetes in NOD mice.

The development of spontaneous autoimmune diabetes in nonobese diabetic (NOD) mice provides for their use as a model of human type 1 diabetes. To test the feasibility of muscle-directed gene therapy to prevent type 1 diabetes, we developed recombinant adeno-associated virus (rAAV) vectors containing murine cDNAs for immunomodulatory cytokines IL-4 or IL-10. Skeletal muscle transduction of female NOD mice with IL-10, but not IL-4, completely abrogated diabetes. rAAV-IL-10 transduction attenuated the production of insulin autoantibodies, quantitatively reduced pancreatic insulitis, maintained islet insulin content, and altered splenocyte cytokine responses to mitogenic stimulation. The beneficial effects were host specific, as adoptive transfer of splenocytes from rAAV IL-10-treated animals rapidly imparted diabetes in naive hosts, and the cells contained no protective immunomodulatory capacity, as defined through adoptive cotransfer analyses. These results indicate the utility for rAAV, a vector with advantages for therapeutic gene delivery, to transfer immunoregulatory cytokines capable of preventing type 1 diabetes. In addition, these studies provide foundational support for the concept of using immunoregulatory agents delivered by rAAV to modulate a variety of disorders associated with deleterious immune responses, including allergic reactions, transplantation rejection, immunodeficiencies, and autoimmune disorders.

Secoisolariciresinol diglucoside from flaxseed delays the development of type 2 diabetes in Zucker rat

Previous research has suggested that type 1 diabetes mellitus may be due to oxidative stress. The role of oxidative stress in type 2 diabetes is not known. Secoisolariciresinol diglucoside (SDG) antioxidant, obtained from flaxseed, has been reported to prevent type 1 diabetes in a rat model. However, its effectiveness in type 2 diabetes is not known. An investigation was made of the effects of SDG isolated from flaxseed (40 mg/kg body wt, orally in drinking water) on the development of diabetes in Zucker diabetic fatty (ZDF)/Gmi-fa/fa female rats, a model of human type 2 diabetes, to determine whether this type of diabetes is due to oxidative stress and whether SDG could prevent the development of diabetes. A total of 10 Zucker lean control and 26 ZDF rats were used in this study. Incidence of diabetes was 100% in untreated and 20% in SDG-treated ZDF rats by the age of 72 days (P < .01). The rats that did not develop diabetes by 72 days of age in the SDG-treated group developed diabetes later on (age 72 to 99 days) except for 10% of the rats that did not develop diabetes for the duration of the study (101 days of age), suggesting that SDG retarded the development of diabetes. Diabetes was associated with an increase in oxidative stress as suggested by an increase in serum malondialdehyde (P < .01). Also, diabetes was associated with an increase in serum total cholesterol and triglycerides (P < .05) and glycated hemoglobin A1C (P < .05). ZDF rats treated with SDG that did not develop diabetes by 70 days of age had no increase in oxidative stress, serum total cholesterol, and glycated hemoglobin. These results suggest that type 2 diabetes is associated with an increase in oxidative stress and that SDG is effective in retarding the development of diabetes. (J Lab Clin Med 2001;138:32-9)

Disease resistant, NOD-related strains reveal checkpoints of immunoregulation in the pancreas

The autoimmune diabetic NOD mouse serves as a model for human type 1 diabetes. Disease development is due to islet beta cell destruction in the context of immune cell infiltration of islets and inflammatory changes throughout the pancreas. In the present study we tried to identify immune reactivity patterns in the pancreas associated with diabetes resistance in NOD-related mouse strains. The pancreata of diabetes-prone female NOD/LtJ, NOD/Bom and of genetically related but diabetes-resistant strains; NOR, NON, NON.NOD-H2g7, NOD.NON-H-2nbl were obtained at the age of 70 days for semiquantitative analysis of insulitis and of mRNA expression by reverse transcriptase PCR. In addition, the response to a single dose of cyclophosphamide for synchronizing and accelerating the progression of insulitis was determined. The progression of insulitis and immune gene expression in response to cyclophosphamide revealed characteristic differences between the six strains. NOD/LtJ and NOD/Bom mice were found significantly to upregulate pancreatic IL-12p40 and IL-18 expression after cyclophosphamide treatment, followed by an increase in IFN-gamma mRNA levels. In contrast, the two MHC-haplotype H-2nbl expressing strains either up-regulated neither IL-12/IL-18 nor IFN-gamma gene expression. The two strains sharing MHC haplotype H-2g7 expression with NOD did respond to cyclophosphamide with IL-12p40/IL-18 gene expression. However, NON.NOD-H-2g7 mice failed to progress to IFN-gamma gene expression. NOR mice progressed to IFN-gamma expression but exhibited sustained IL-4 gene expression. Only severe intra-insulitis was associated with the expression of inducible NO synthase. The comparison of diabetes-prone and diabetes-resistant strains revealed three checkpoints of immune regulation in the pancreas. The earliest checkpoint is the induction of an IL-12p40/IL-18 response in innate immune or antigen-presenting cells. The next level of control is at the induction of IFN-gamma gene expression, and a third checkpoint is the maintenance or loss of antagonistic Th2 type reactions.

Oxidative stress as a mechanism of diabetes in diabetic BB prone rats: effect of secoisolariciresinol diglucoside (SDG).

Secoisolariciresinol diglucoside (SDG) isolated from flaxseed has antioxidant activity and has been shown to prevent hypercholesterolemic atherosclerosis. An investigation was made of the effects of SDG on the development of diabetes in diabetic prone BioBreeding rats (BBdp rats), a model of human type I diabetes [insulin dependent diabetes mellitus (IDDM)] to determine if this type of diabetes is due to oxidative stress and if SDG can prevent the incidence of diabetes. The rats were divided into three groups: Group I, BioBreeding normal rats (BBn rats) (n = 10); group II, BBdp untreated (n = 11); and group III, BBdp treated with SDG 22 mg/kg body wt, orally) (n = 14). Oxidative stress was determined by measuring lipid peroxidation product malondialdehyde (MDA) an index of level of reactive oxygen species in blood and pancreas; and pancreatic chemiluminescence (Pancreatic-CL), a measure of antioxidant reserve. Incidence of diabetes was 72.7% in untreated and 21.4% in SDG-treated group as determined by glycosuria and hyperglycemia. SDG prevented the development of diabetes by approximately 71%. Development of diabetes was associated with an increase in serum and pancreatic MDA and a decrease in antioxidant reserve. Prevention in development of diabetes by SDG was associated with a decrease in serum and pancreatic-MDA and an increase in antioxidant reserve. These results suggest that IDDM is mediated through oxidative stress and that SDG prevents the development of diabetes.

Gastrointestinal endocrine cells in an animal model for human type 2 diabetes.

Endocrine cells were investigated in various parts of the gastrointestinal tract in an animal model for human type 2 diabetes, namely the homozygous obese mouse. As controls, age- and sex-matched homozygous lean mice were used. The different endocrine cell types were stained by using immunocytochemistry and quantified by computerized image analysis. The numbers of PYY- and enteroglucagon-immunoreactive (IR) cells were decreased in the colon of obese diabetic mice vis-à-vis controls. Serotonin-IR cells were significantly decreased in numbers in the duodenum and colon. Furthermore, colonic serotonin cells had a high cell secretory index (CSI), but an unchanged nuclear area, indicating an impaired cellular release of this amine. There was no statistical difference between obese diabetic mice and lean controls regarding the numbers and CSI of antral gastrin/CCK-, somatostatin- and serotonin-; and duodenal secretin-, gastric inhibitory peptide (GIP)-, CCK/gastrin-, and somatostatin-IR cells; nor was there any difference regarding nuclear area, with the exception of the antral somatostatin- and duodenal GIP-IR cells. It is concluded that the abnormalities in gut endocrine cells observed in the present study might explain the gut dysmotility reported in animal models for human diabetes. They may also be of importance in gastrointestinal dysfunction occurring in diabetes patients.

β-Cell Apoptosis in an Accelerated Model of Autoimmune Diabetes

The non-obese diabetic (NOD) mouse is a model of human type 1 diabetes in which autoreactive T cells mediate destruction of pancreatic islet beta cells. Although known to be triggered by cytotoxic T cells, apoptosis has not been unequivocally localized to beta cells in spontaneously diabetic NOD mice. We created a model of accelerated beta-cell destruction mediated by T cells from spontaneously diabetic NOD mice to facilitate the direct detection of apoptosis in beta cells. NOD.scid (severe combined immunodeficiency) mice were crossed with bm1 mice transgenically expressing the costimulatory molecule B7-1 (CD80) in their beta cells, to generate B7-1 NOD.scid mice. Apoptosis in islet cells was measured as DNA strand breakage by the TdT-mediated-dUTP-nick end labeling (TUNEL) technique. Adoptive transfer of splenocytes from spontaneously diabetic NOD mice into B7-1 NOD.scid mice caused diabetes in recipients within 12-16 days. Mononuclear cell infiltration and apoptosis were significantly greater in the islets of B7-1 NOD.scid mice than in nontransgenic NOD.scid mice. Dual immunolabeling for TUNEL and either B-7 or insulin, or the T cell markers CD4 and CD8, and colocalization by confocal microscopy clearly demonstrated apoptosis in beta cells as well in a relatively larger number of infiltrating T cells. The clearance time of apoptotic beta cells was estimated to be less than 6 min. B7-1 transgenic beta cells undergo apoptosis during their accelerated destruction in response to NOD mouse effector T cells. Rapid clearance implies that beta cells undergoing apoptosis would be detected only rarely during more protracted disease in spontaneously diabetic NOD mice.

T cells with multiple fine specificities are used by non-obese diabetic (NOD) mice in the response to GAD(524-543).

Insulin-dependent diabetes mellitus (IDDM) in the non-obese diabetic (NOD) mouse results from a T lymphocyte mediated destruction of the insulin-producing beta cells of the pancreas and serves as a model for human type I diabetes. The NOD mouse develops insulitis at 4 weeks of age and diabetes later in life. It has previously been shown that a T helper 1 (Th1) response to the islet antigen, glutamic acid decarboxylase (GAD65, henceforth GAD) spontaneously develops in NOD mice concurrent with the onset of lymphocytic infiltration into the islets (insulitis). The proliferative T cell response in the spleen is initially confined to the carboxy-terminal region of GAD65 (peptides 509-528 and 524-543) followed by a progression to nearby determinants and a variety of upstream determinants. We have produced a set of overlapping synthetic peptides spanning the 509-543 region of GAD and surveyed the responses raised by immunization with peptide GAD(524-543), which is the more immunogenic of the two peptides. NOD mice immunized with GAD(524-543) demonstrate splenic proliferative responses to 524-538 and 527-541 but not to 521-535 or 530-543. Four T cell hybridomas were produced from spleen cells of GAD(524-543)-immunized NOD female mice. Each hybridoma displayed a unique cytokine profile when stimulated with peptides 524-538 and 527-541, assaying IL-2, IFN-gamma, and IL-5 production by peptide-stimulated hybridomas. To identify MHC and TCR contact residues critical for the stimulation of the hybridomas, a truncated peptide (GAD 526-538) and a panel of analogue peptides were synthesized containing single-amino acid substitutions. Hybridoma 35.13.2 was non-responsive to the truncated peptide and all of its variants. However, the four residues 530 (A), 531 (P), 536 (R), and 537 (M) were found to be critical for the activation of the three remaining hybridomas, suggesting that these positions in the GAD-524-543 determinant were MHC binding residues or conserved TCR contact sites.

GAD65 is recognized by T-cells, but not by antibodies from NOD-mice.

Since the 64kDa-protein glutamic acid decarboxylase (GAD) is one of the major autoantigens in T-cell mediated Type 1 diabetes, its relevance as a T-cell antigen needs to be clarified. After isolation of splenic T-cells from non-obese diabetic (NOD) mice, a useful model for human Type 1 diabetes, we found that these T-cells proliferate spontaneously when incubated with human GAD65, but only marginally after incubation with GAD67, both recombinated in the baculovirus system. No effect was observed with non-diabetic NOD mice or with T-cells from H-2 identical NON-NOD-H-2g7 control mice. It has been published previously that NOD mice develop autoantibodies against a 64kDa protein detected with mouse beta cells. In immunoprecipitation experiments with sera from the same NOD mice and 35S-methionine-labelled GAD, no autoantibody binding could be detected. We conclude firstly that GAD65 is an important T-cell antigen which is relevant early in the development of Type 1 diabetes and secondly that there is an antigenic epitope in the human GAD65 molecule recognized by NOD T-cells, but not by NOD autoantibodies precipitating conformational epitopes. Our results therefore provide further evidence that GAD65 is a T-cell antigen in NOD mice, being possibly also involved in very early processes leading to the development of human Type 1 diabetes.

Characterization of giant perivascular spaces in the thymus of the nonobese diabetic mouse.

The nonobese diabetic (NOD) mouse spontaneously develops insulin-dependent diabetes mellitus as a result of autoimmune destruction of β-cells in the pancreatic islets of Langerhans. It is thus widely accepted as a relevant experimental model for human type I diabetes 1–3. Clinically-defined disease occurs more predominantly in females and can be observed from the age of 3 months. Insulitis always precedes the onset of diabetes, but appears in both males and females from the age of 3–4 weeks4–5.

The role of insulin-like growth factors in diabetic kidney disease.

Early renal manifestations of type I diabetes include kidney enlargement, increased glomerular filtration rate, and renal plasma flow. These hemodynamic changes may be caused by a number of factors, including growth hormone and/or insulin-like growth factor-I (IGF-I). Streptozotocin-induced insulinopenic diabetes in rats represents a model of human type I diabetes and is associated with the early hemodynamic changes in the kidney seen in poorly controlled type I diabetic patients. These changes are preceded by an accumulation of IGF-I peptide in the kidney. Insulin-like growth factor-I is not locally produced, but rather accumulates from circulating IGF-I, trapped by increased levels of IGF-binding proteins, particularly IGF-binding protein-1. The hemodynamic effects, reproduced by infusions of recombinant human IGF-I in normal rats, may be blocked by co-infusion of a kinin-receptor antagonist, suggesting that at least one of the mechanisms involved is the kallikrein-kinin system. These studies strongly support the notion that the IGF system may play a role in early hemodynamic manifestations of the diabetic kidney. Whether these effects lead to long-term diabetic renal disease remains to be studied.

Evidence for antigen driven selection in two monoclonal auto-antibodies derived from nonobese diabetic mice

The nonobese diabetic (NOD) mouse is a model of human type I diabetes. This diabetes is due to massive infiltration of the pancreatic β cell of islets by autoreactive T cells (insulitis) followed by the destruction of insulin-producing cells. Circulating autoantibodies are also detected, notably against glutamic acid decarboxylase, peripherin and insulin. Two monoclonal autoantibodies directed against insulin and peripherin were obtained by fusing NOD spleen and myeloma cells. We report here the nucleotide sequence of the genes encoding for the V regions of these two antibodies. Somatic mutations were identified by comparing the light chain nucleotide sequence of one of these autoantibodies with its germline counterpart precursor established from NOD mice after PCR gene amplification. The other one displays N additions on both sides of the D region. These results strongly suggest that both autoantibodies have undergone diversification, either N additions or somatic mutations, and therefore present structural features of antibodies derived from animals immunized against exogenous antigens.

RFLP analysis of the MHC class III region defines unique haplotypes for the non-obese diabetic, cataract Shionogi and the non-obese non-diabetic mouse strains

The non-obese diabetic (NOD) mouse strain which spontaneously develops diabetes is a model for human Type 1 (insulin-dependent) diabetes mellitus. At least one of several genes controlling diabetes in the NOD mouse has been mapped to the MHC. Although previous experiments have implicated the MHC class II genes in the development of the disease, the existence of other MHC linked susceptibility genes has not been ruled out. In order to identify these susceptibility genes we have further characterized the MHC haplotype of the NOD mouse and two non-diabetic sister strains, the non-obese non-diabetic (NON) and cataract Shionogi (CTS). We have examined the mouse MHC class III region for the presence of homologous genes to 17 newly isolated human MHC class III region genes (G1, G2, G4, G6, G7a/valyl-tRNA synthetase, HSP70, G8, G9, G10, G12, G13, G14, G15, G16, G17 and G18). We detect unique hybridizing DNA fragments for 16 of the 17 genes in six inbred mouse strains (NOD, NON, CTS, B10, BALB/c and CBA/J) indicating that this part of the H-2 region is similar to the human MHC class III region. Using a panel of restriction enzymes we have defined RFLPs for 6 (G2, G6, HSP70, G12, G16, G18) of the 16 cross-hybridizing probes. The RFLPs demonstrate that NOD, NON and CTS mouse strains each have a distinct MHC haplotype in the MHC class III region.