Insulitis In Non-obese Diabetic Mice Research Articles

Synthesis and kinetics of cyclization of MHC class II-derived cyclic peptide vaccine for diabetes.

Conformationally constrained cyclic peptides are known to be better vaccines because of their ability to mimic the native structure of a protein against which an immune response is sought. To test the hypothesis of using conformationally constrained, disease-associated, MHC-derived peptides as vaccines for the prevention of type I diabetes, a 22 amino acid nonobese diabetic(NOD) mouse MHC class II-derived synthetic peptide was cyclized by the formation of end-to-end disulfide bonds and used to prevent diabetes and insulitis in NOD mice. The peptide was synthesized by Fmoc chemistry and cyclized using two methods: a commercially available cyclizing resin (Ekathiox) and air oxidation. When a 10 m excess of resin was used, the Ekathiox yielded a substantial amount of cyclic peptide with few or no side reactions. The kinetics of cyclization by air oxidation at different temperatures indicated that increasing both temperature and pH decreased the cyclization time significantly. Air oxidation at pH 10 at 37-55 degrees C yielded the desired product within 2 h.

A single initiating autoantigen for diabetes?

Insulin-dependent diabetes mellitus (IDDM) results from the T cell-mediated destruction of the insulin-producing β-cells. Intensive efforts have been directed towards understanding the events that trigger β-cell autoreactivity. Previous studies in an animal model of IDDM, the nonobese diabetic (NOD) mouse, have shown that T-cell reactivity first arises against glutamate decarboxylase (GAD) and then spreads to other β-cell antigens 1 Kaufman D.L. et al. Spontaneous loss of T-cell tolerance to glutamic acid decarboxylase in murine insulin-dependent diabetes. Nature. 1993; 366: 69-72 Crossref PubMed Scopus (1045) Google Scholar , 2 Tisch R. et al. Immune response to glutamic acid decarboxylase correlates with insulitis in non-obese diabetic mice. Nature. 1993; 366: 72-75 Crossref PubMed Scopus (938) Google Scholar . The early inactivation of GAD-reactive T cells could prevent the development of autoreactivity, suggesting that the development of T-cell reactivity against GAD is key to the initiation and propagation of autoimmune responses against β-cells.

The role of Fas ligand in the development of insulitis in nonobese diabetic mice.

The role of Fas ligand-induced lymphocyte apoptosis in the development of insulitis was studied in nonobese diabetic (NOD) mice. Fas ligand with a molecular weight of 45 kD appeared in the pancreas of NOD mice during the onset of insulitis as demonstrated by western blot analysis. In situ DNA end-labeling (ISEL) of the pancreases of NOD mice demonstrated positive cells in the islets of Langerhans, with an age-dependent increase in the density and frequency of animals with islets containing ISEL-positive cells. In the pancreatic islets of BALB/c mice, no ISEL-positive cells were observed in any of the age groups studied. In ultrastructural analysis degenerating cells with condensed or fragmented nuclei and plasma membranes detached from neighboring cells were observed both in and around the islets. In some cases, these cells were being phagocytosed by the neighboring islet cells. Degenerating cells with characteristics of lymphocytes were seen in contact with healthy lymphocytes around the islets. Neutralizing Fas ligand antibodies affected 3H-thymidine incorporation of CD3+CD28+ pancreatic lymphocytes from 12- to 30-week-old NOD mice in one of three cultures. There was no difference in the effect of neutralizing Fas ligand antibodies between pancreatic and blood lymphocytes. The present results on an increase in density of apoptotic cells in pancreatic islets of NOD mice simultaneously with the onset of insulitis and appearance of Fas ligand suggest that the pancreas infiltrating lymphocytes may be destroyed by Fas ligand-induced apoptosis.

Administration of IL-4 Prevents Autoimmune Diabetes but Enhances Pancreatic Insulitis in NOD Mice

The present study demonstrated that the administration of recombinant interleukin-4 (rIL-4) prevented overt diabetes in nonobese diabetic (NOD) mice whose T cells produced relatively low amounts of IL-4. However, massive insulitis was observed in rIL-4-treated NOD mice. The flow cytometric analysis of islet-infiltrating T cells revealed that the number of CD45RB lowCD4 +T cells was significantly increased by in vivoadministration of rIL-4. By measuring the cytokine production of splenic T cells after stimulation, it was shown that CD45RB lowCD4 +T cells predominantly produced IL-4 and IL-10 but produced less IL-2 and interferon-γ (IFN-γ). A semiquantitative reverse-transcriptase polymerase chain reaction assay revealed a higher expression of IL-4 and IL-10 mRNA and an apparent decrease in IFN-γ mRNA in the islets of NOD mice which were administered rIL-4. These results suggested that autoreactive CD45RB lowCD4 +T helper 2 (Th2)-like cells which developed following rIL-4 administration were predominant in the infiltrate of the islets, and overt diabetes was prevented. On the other hand, when splenocytes from rIL-4-treated NOD mice were transferred to irradiated NOD recipients, along with splenocytes from diabetic NOD mice, all of the recipient mice became diabetic within 8 weeks after transfer. Considered together, a supplement of rIL-4 administered to NOD mice may protect against autoimmune diabetes by facilitating the development of Th2-like autoreactive T cells in the islets.

Alleviation of insulitis in NOD mice is associated with expression of transgenic MHC E molecules on primary antigen-presenting cells.

Major histocompatibility complex (MHC) class II genes are important in the pathogenesis of insulin-dependent diabetes mellitus (IDDM) both in the mouse and in man. The non-obese diabetic (NOD) mouse, which is a good model for human IDDM, has a particular MHC class II with an A complex consisting of A alpha d and the unique A beta g7 chain, as well as an absent E molecule due to a deletion in the Ea promoter region. Transgenic insertion of a functional Ea gene protects against insulitis and diabetes, but when the transgene expression is restricted to certain compartments of the immune system by deleting parts of the promoter region, the protection against insulitis is disrupted. We have analysed three promoter-mutated lines where one lacks expression on B cells and has a reduced expression on approximately 1/3 of the dendritic cells and macrophages (Sma), one lacks thymic cortical expression and has a slightly reduced B-cell expression (delta X), and one lacks expression in the thymic medulla, on macrophages, dendritic cells and about half of the B cells (delta Y). None of these lines is protected against insulitis, but Sma and delta X display a reduced intensity of insulitis, with an average of 10-15% of the islets infiltrated in each mouse, while delta Y resembles non-transgenic mice with 30-35% infiltrated islets. Bone-marrow chimeras between Sma and delta Y mice demonstrate that peripheral cells of Sma origin reduce insulitis significantly when developed in the delta Y host, while insulitis is enhanced when delta Y bone marrow is given to Sma mice. This shows that E expression on the primary antigen-presenting macrophages and dendritic cells is of crucial importance to the alleviation of insulitis.

IL-4 expression at the onset of islet inflammation predicts nondestructive insulitis in nonobese diabetic mice.

In nonobese diabetic mice, autoimmune diabetes progresses in an age-linked and gender-dependent manner. Insulitis begins in male and female mice at approximately 1 mo of age; however, 70 to 90% of females, but only 10 to 20% of males, become diabetic by 6 mo. Multiple studies propose that proinflammatory Th1 and immunomodulatory Th2 cytokines impact diabetes pathogenesis, but the role of these cytokines in spontaneous diabetes progression is not yet clear. We used quantitative reverse-transcriptase-coupled PCR to analyze expression of cytokines and APC costimulatory molecules in the islets of 20- to 180-day-old male and female nonobese diabetic littermates, and identified three stages in diabetes progression. At 1 to 2 mo of age, islet-infiltrating T cells displayed a Th1 cytokine bias in females, and a Th2 cytokine bias in males. In females, stage II (2-3 mo of age) was characterized by an increase in islet-infiltrating T cells, APC, and Th1 cytokines, whereas male infiltrates did not increase in size, and Th1 cytokine expression continued to decline during this interval. Islet infiltration reached a plateau (stage III) in 3- to 4-mo-old females, months before overt diabetes onset. Our data imply that Th cytokine expression in early insulitis exerts substantial impact on beta cell destruction and overt diabetes. A clinical implication of our results is that young individuals in the early stages of insulitis are ideal candidates for therapeutic intervention to minimize beta cell destruction and morbidity.

Mixed allogeneic chimerism induced by a sublethal approach prevents autoimmune diabetes and reverses insulitis in nonobese diabetic (NOD) mice.

Evidence in experimental models suggests that many autoimmune diseases can be prevented by transplantation of bone marrow from disease-resistant donors. For potential clinical application, it would be important to avoid the morbidity and mortality associated with lethal conditioning and achieve mixed chimerism using less than complete recipient ablation. We report here for the first time that stable chimerism achieved in NOD mice using a sublethal radiation-based conditioning approach is sufficient to prevent beta-cell destruction and abrogate insulitis in prediabetic NOD mice. The percentage of NOD mouse recipients (8 wk of age) that engrafted with donor bone marrow correlated with the dose of irradiation and number of bone marrow cells transplanted. Engraftment of B10.BR bone marrow occurred in > or = 94% of animals receiving > or = 750 cGy of total body irradiation before bone marrow transplantation and > or = 30 x 10(6) bone marrow cells, while reproducible engraftment did not occur at radiation doses of less than 700 cGy and cellular doses of less than 30 x 10(6) bone marrow cells. All chimeric animals remained free of diabetes (n = 38) for 10 mo following bone marrow transplantation. Moreover, in all animals examined, no insulitis was present from 12 to 36 wk following reconstitution. In striking contrast, 61% (22 of 36) of NOD recipients that were conditioned but did not receive bone marrow developed acute diabetes by 12 mo. Insulitis was present in all remaining animals. These results suggest that allogeneic chimerism achieved using a sublethal conditioning approach can prevent the onset of diabetes and even reverse preexisting insulitis in NOD mice.

Protection from experimental autoimmune diabetes in the non-obese diabetic mouse with soluble interleukin-1 receptor.

We have evaluated the effects of a treatment with soluble interleukin-1 receptor (sIL-1R) in the accelerated model of autoimmune diabetes induced by cyclophosphamide (CY) in the non-obese diabetic (NOD) mouse. Prior to the CY challenge (350 mgkg body weight), female euglycemic NOD mice were randomly divided into three groups (A-C). Groups B and C were treated daily from 1 day before to 13 days after the CY challenge with sIL-1R at doses of 0.2 and 2 mg/kg body weight. Group A was treated with PBS. By 2 weeks after CY administration, an acute form of autoimmune diabetes with glycosuria, hyperglycemia and severe insulitis occurred in the majority (13/20, 65%) of the control mice (group A). In contrast, repeated injections with sIL-1R protected NOD mice from insulin-dependent diabetes mellitus (IDDM) development in a dose-dependent fashion; the incidence of IDDM was 53.3% (8/15) in the mice treated with 0.2 mg/kg and only 6.7% (1/15) in those treated with 2 mg/kg. However, none of the doses of the sIL-1R reduced the extent of insulitis in NOD mice. Importantly, the anti-diabetogenic property of sIL-1R may not involve major T cell function impairment; accordingly, in parallel experiments, splenic lymphoid cells from NOD mice not challenged with CY, but treated with 2 mg/kg sIL-1R for 5 consecutive days showed a normal distribution of mononuclear cell subsets and maintained their capacity to secrete interferon-gamma and IL-2 and to proliferate in response to polyclonal mitogenic stimulation with concanavalin A.

Immune response to glutamic acid decarboxylase correlates with insulitis in non-obese diabetic mice.

Immune response to glutamic acid decarboxylase correlates with insulitis in non-obese diabetic mice.

Reactive oxygen intermediates in autoimmune islet cell destruction of the NOD mouse induced by peritoneal exudate cells (rich in macrophages) but not T cells.

The non-obese diabetic (NOD) mouse spontaneously develops autoimmune Type 1 (insulin-dependent) diabetes mellitus. NOD mice exhibit massive infiltrates of T cells and macrophages into pancreatic islets (insulitis) prior to diabetes. The contribution of oxygen free radicals to the development of insulitis in NOD mice was examined by administration of its scavengers, such as superoxide dismutase and catalase. Bovine superoxide dismutase and catalase were each coupled to polyethylene glycol. The treatment with superoxide dismutase-polyethylene glycol reduced the number of islets with insulitis and increased the undamaged islet tissue, as compared with the control group. The treatment with catalase-polyethylene glycol showed a similar tendency which did not reach significance. Using a flow cytometric assay of the oxidation of 2', 7'-dichlorofluorescein, the content of reactive oxygen intermediates in islet cells in the culture system was measured and the effect of peritoneal exudate cells and T cells on their production examined. Peritoneal exudate cells, but not T cells, from NOD mice increased the content of reactive oxygen intermediates in islet cells of either the NOD mouse or the ILI mouse (MHC-identical to NOD); the addition of superoxide dismutase to the culture medium suppressed this increase in NOD or ILI islet cells. The present data support the concept that production of oxygen free radicals mediated by macrophages can damage islet beta cells, directly resulting in autoimmune Type 1 diabetes in NOD mice.

Effect of lipoic acid on cyclophosphamide-induced diabetes and insulitis in non-obese diabetic mice

In an animal model of type I diabetes, the non-obese diabetic (NOD) mouse, the influence of the antioxidant lipoic acid (LA) on the development of diabetes was investigated. Acceleration of diabetes development with cyclophosphamide (CY) resulted in 60% diabetic animals with severely infiltrated islets within 1–3 weeks. Daily administration of lipoic acid for 20 or 30 days around cyclophosphamide treatment suppressed the incidence of diabetes to 30% ( P<0.05) and 33%, respectively. Semiquantitative analysis of islet infiltration showed a reduction of severe intraislet infiltration and an increase in the percentage of islets with mild peri-insular and periductular infiltrates (from 8.4 to 29.6 and 25.9%, respectively, P<0.01) after lipoic acid treatment. These results show that the protective effect of lipoic acid on diabetes development correlates with partial suppression of islet inflammation. The anti-inflammatory action of lipoic acid may be due to its ability to scavenge oxygen radicals and to suppress nitric oxide production.

Immune response to glutamic acid decarboxylase correlates with insulitis in non-obese diabetic mice.

Knowing the autoantigen target(s) in an organ-specific autoimmune disease is essential to understanding its pathogenesis. Insulin-dependent diabetes mellitus (IDDM) is an autoimmune disease characterized by lymphocytic infiltration of the islets of Langerhans (insulitis) and destruction of insulin-secreting pancreatic beta-cells. Several beta-cell proteins have been identified as autoantigens, but their importance in the diabetogenic process is not known. The non-obese diabetic (NOD) mouse is a murine model for spontaneous IDDM. Here we determine the temporal sequence of T-cell and antibody responses in NOD mice to a panel of five murine beta-cell antigens and find that antibody and T-cell responses specific for the two isoforms of glutamic acid decarboxylase (GAD) are first detected in 4-week-old NOD mice. This GAD-specific reactivity coincides with the earliest detectable response to an islet extract, and with the onset of insulitis. Furthermore, NOD mice receiving intrathymic injections of GAD65 exhibit markedly reduced T-cell proliferative responses to GAD and to the rest of the panel, in addition to remaining free of diabetes. These results indicate that the spontaneous response to beta-cell antigens arises very early in life and that the anti-GAD immune response has a critical role in the disease process during this period.

Vascular addressins are induced on islet vessels during insulitis in nonobese diabetic mice and are involved in lymphoid cell binding to islet endothelium.

In the nonobese diabetic (NOD) mouse, lymphocytic and monocytic infiltration of the pancreatic islets leads to beta cell destruction. To investigate the mechanisms by which lymphocytes enter the NOD pancreas, pancreata were immunostained using monoclonal antibodies to a variety of adhesion molecules known to be involved in lymphocyte binding to vascular endothelium, an initial step in the migration of lymphocytes from blood into organized lymphoid and inflamed tissues. These adhesion molecules include: lymphocyte homing receptors involved in tissue-selective binding of lymphocytes to peripheral lymph node (L-selectin) or mucosal lymphoid tissue (LPAM-1, alpha 4 beta 7-integrin) high-endothelial venules (HEV); and HEV ligands peripheral vascular addressin (PNAd) and mucosal vascular addressin (MAdCAM-1). In NOD pancreata, alpha 4 beta 7 is expressed on most infiltrating cells at all stages of insulitis, whereas L-selectin expression is more pronounced on cells in the islets at later stages. During the development of insulitis, MAdCAM-1 and to a lesser extent PNAd became detectable on vascular endothelium adjacent to and within the inflamed islets. The Stamper-Woodruff in vitro assay was used to examine lymphoid cell binding to such vessels. These functional assays show that both the mucosal (MAdCAM-1/alpha 4 beta 7) and the peripheral (PNAd/L-selectin) recognition systems are involved in this binding. Our findings demonstrate that expression of peripheral and mucosal vascular addressins is induced on endothelium in inflamed islets in NOD pancreas, and that these addressins participate in binding lymphoid cells via their homing receptors. This suggests that these adhesion molecules play a role in the pathogenesis of diabetes in these mice by being involved in the migration of lymphocytes from blood into the inflamed pancreas.

Limited heterogeneity of T-cell receptor Vβ gene expression in the early stage of insulitis in NOD mice

Non-obese-diabetic (NOD) mice spontaneously develop type I diabetes. The disease starts with T cells infiltrating the islets of Langerhans. We therefore examined the T-cell receptor (TCR) Vβ region repertoire in islet infiltrates from individual female NOD mice from 4 to 10 week old by polymerase chain reaction (PCR) using Vβ1-Vβ17 specific oligonucleotides. The study revealed a limited heterogeneity of TCR Vβ transcripts with a predominance of Vβ1 at the onset of insulitis, i.e. at 4 weeks of age. The TCR VDJβ sequences of the Vβ1 PCR fragments were identical in most of the individual mice. Among several different mice, similarities in the β-junctional regions were detected. In contrast, a large heterogeneity of TCR Vβ usage was found in mice with advanced insulitis, i.e., from 6 weeks of age on. Thus, these data suggest a limited heterogeneity of TCR Vβ usage with a predominance of Vβ1 at the initiation phase of the disease.

Prevention of Diabetes but not Insulitis in NOD Mice Injected with Antibody to CD4

Non-obese diabetic (NOD) mice were injected with a rat monoclonal antibody to CD4 from birth every two weeks through 6 months of age. These animals gained weight normally but < 11% of their spleen T cells were CD4+, compared with 28% of CD4+ in controls injected with polyclonal rat IgG. The reduction in CD4 cell percentage was associated with a reduction in the number of cells in the thymus and spleen following the injection. CD4+ cells which survived the injections were nevertheless able to enter cell cycle when stimulated by Con A. None of the CD4-treated NOD mice became diabetic by 6 months of age and none of the animals studied histologically at this time had insulitis. At 9 months of age (three months after stopping the CD4 injections) the mice made antibody to human IgG. At 1 year of age most of the male mice had insulitis, although none of the male or female mice had become spontaneously diabetic. Two thirds of animals injected with cyclophosphamide at 16 months became diabetic within 3 weeks. The results confirm that treatment with CD4 antibody in the first 6 months suffices to reduce the incidence of diabetes in NOD mice. The treatment does not prevent the subsequent development of insulitis in injected mice and does not prevent the accumulation of cells capable of causing overt diabetes after cyclophosphamide injection.

Prevention of insulitis and diabetes in nonobese diabetic mice by administration of FK506.

We investigated the preventive effect of the immunosuppressive agent FK506 on autoimmune insulitis in nonobese diabetic mice. The mice were given FK506 in a dose of 1.0 mg/kg, every other day, from age 2 to 12, 2 to 6, and 4 to 12 weeks, respectively; after which, the incidence of insulitis and overt diabetes was monitored. Effects of FK506 on immune reactions to beta cells were also investigated by using both syngeneic and allogeneic islet transplants. Treatment with FK506 in mice from age 2 weeks prevented completely the onset of overt diabetes, and the incidence of insulitis was reduced to less than 10% at age 30 weeks. Treatment of mice with FK506 from age 4 weeks was less effective in preventing insulitis and the onset of diabetes. In case of islet transplantation, FK506 treatment of NOD mice from age 2 to 6 weeks prevented autoimmune responses both in syngeneic islets and in allogeneic islets, which share the same H-2 antigen with the nonobese diabetic mouse. These results also indicate that the recognition of islet antigens and the generation of autoimmune-reactive T lymphocytes start between 2 and 4 weeks of age, and FK506 prevents an autoimmune reaction.

Prevention of autoimmune insulitis in nonobese diabetic mice by expression of major histocompatibility complex class I Ld molecules.

Nonobese diabetic (NOD) mice spontaneously develop a T-cell-mediated autoimmune disease that is similar in many respects to insulin-dependent diabetes mellitus in humans. NOD mice were shown to express major histocompatibility complex class I Kd and Db antigens. To examine the possible involvement of major histocompatibility complex class I molecules in the development of autoimmune insulitis, we attempted to express a different type of class I molecule in NOD mice by crossing C57BL/6 mice transgenic for the class I Ld gene with NOD mice. The backcross progeny expressed the Ld antigen on the peripheral blood lymphocytes at a level comparable with that of the BALB/c mice. The cell surface expression of endogenous class I and class II antigens on the peripheral blood lymphocytes was not affected. Analysis of these mice revealed that the expression of the class I Ld antigen significantly reduced the incidence of insulitis at 20 weeks of age. In situ hybridization of a biotinylated probe on mouse chromosomes showed that the Ld transgene was located in the E area of chromosome 6 with which no genetic linkage to insulin-dependent diabetes mellitus was demonstrated. These results suggest that the NOD-type class I molecules are involved in the development of insulitis in NOD mice.

Monoclonal anti-tumor necrosis factor antibody renders non-obese diabetic mice hypersensitive to irradiation and enhances insulitis development.

In attempt to evaluate biological roles of tumor necrosis factor (TNF), we studied the effects of anti-TNF mAb in non-obese diabetic (NOD) mice. Anti-murine TNF mAb rendered NOD mice hypersensitive to the lethal effects of radiation and prevented the reconstitution of lethally irradiated mice with adoptively transferred lymphocytes. While TNF-alpha reduced the incidence of diabetes development in the adoptive transfer system even when given 6 days post-transfer, mAb to TNF could not reduce or increase the incidence of diabetes compared to control mice. Administration of TNF-alpha for 4 or 8 weeks significantly reduced the incidence of spontaneous insulitis in NOD mice, while anti-TNF mAb given for 8 weeks increased the incidence of insulitis significantly.

Overexpression of class I major histocompatibility complex accompanies insulitis in the non-obese diabetic mouse and is prevented by anti-interferon-? antibody

Overexpression of class I major histocompatibility complex (MHC) proteins on pancreatic islet cells is a characteristic of autoimmune Type 1 (insulin-dependent) diabetes mellitus in humans and in animal models. Studies of post-mortem pancreases from humans with Type 1 diabetes suggest that overexpression of class I MHC proteins may precede mononuclear cell infiltration of the islets (insulitis). Pancreatic histology from the earliest stages of human Type 1 diabetes is rarely available. We have used the non-obese diabetic mouse, given cyclophosphamide to accelerate Beta-cell destruction, to investigate the temporal relationship between the overexpression of class I MHC protein and mRNA and other pathological changes associated with Beta-cell destruction. Prior to cyclophosphamide, immunoperoxidase staining showed that expression of class I MHC proteins was greater on islet cells and infiltrating inflammatory cells of the non-obese diabetic mouse than on islet cells of other mouse strains, whereas staining on exocrine cells was similar. On day three after cyclophosphamide administration, when insulitis had regressed, islet class I MHC protein expression had diminished. A dramatic increase in class I MHC protein expression occurred between days seven and nine, concomitant with reinfiltration of the islets by mononuclear cells; overexpression was seen both on islet cells and on surrounding exocrine cells, but only in the presence of mononuclear cell infiltration. By day 21, class I MHC protein overexpression was again confined to the islets, the exocrine pancreas being free of infiltration. Class I mRNA also increased dramatically by day eight but had virtually returned to normal by day 12.2+ effected by cytokines secreted by activated immuno-inflammatory cells. Class I MHC overexpression should enhance targeting of cytotoxic T cells to Beta cells bearing autoantigen.

Transgenic Mouse as a Tool in Human Genetics.

Transgenic mice have been used for analyses of cis-acting elements which is involved in the tissue-specific and developmental-specific expression, for analyses of physiological function of genes, or for the production of human disease model. This approach is especially successful in the field of Genetics and Immunology. For the past several years we have been studying one of human genetic diseases, Familial amyloidotic polyneuropathy (FAP) . FAP is one type of systemic amyloidosis and is an autosomal dominant disorder characterized by the extracellular deposition of amyloid fibrils and by prominent peripheral and autonomic nerve involvement. Accumulating evidences suggest that the main cause of this disease is the presence of the mutant transthyretin (TTR) gene. However, many questions in relation to the disease process remain to be elucidated. In addition, there is no effective therapy for FAP. To elucidate the pathological process of this disease development and to devise a new method for treatment, we have produced a transgenic mouse model of FAP by introducing the human mutant transthyretin (hMet30) gene. In these transgenic mice, amyloid deposition starts to occur at around 6 months of age and the amount of amyloid deposition increases gradually with aging. Amyloid deposition is observed in many tissues including heart, kidney and thyroid gland, where amyloid deposition is commonly observed in FAP patient. There was a big variation at the age of onset although we used an inbred strain of mouse, C57BL/6, in these experiments and the serum concentrations of hMet30 were about the same in these transgenic mice. The hMet30 gene was found to be expressed from fetal stage. These results suggest that amyloid deposition itself starts to occur late in life and that this amyloid deposition is influenced by environmental factors.Several years ago we showed that the major histocompatibility complex (MHC) class II gene is identical to the immune response gene by demonstrating that the immune response can be restored by the new expression of class II molecules on immunocompetent cells. Recent evidence suggest that class II molecule is involved in the generation of autoimmune disease, such as insulin dependent diabetes mellitus (IDDM) . NOD (non-obese diabetic) mouse is shown to be a mouse model for human IDDM. Concerning the class II genes, NOD mouse has two characteristic features, the lack of I-E and the presence of unique I-A. In order to examine the role of these class II molecules, we produced NOD transgenic mice by introducing murine class II genes. The incidences of insulitis in I-E and I-A transgenic mice were much lower than that of control NOD mice suggesting that both the lack of I-E and the presence of unique I-A are responsible for the development of insulitis in NOD mouse. Our data also suggest that the amino acid at position 57 of Aβ chain is not the only cause for insulitis.Recent technical advance in gene targeting together with the conventional transgenic tecnnique will be a powerful tool not only for the establishment of a transgenic mouse model for the human disease but also for analyses of disease process of human disease.