Diabetes segregates as a single locus in crosses between inbred BB rats prone or resistant to diabetes.

Diabetes-prone (DP) BB rats spontaneously develop insulin-dependent diabetes resembling type 1 diabetes mellitus in man. They also exhibit lifelong T cell deficiency. The segregation of both diabetes and lymphopenia was studied in crosses between this inbred line of rats and the related but nondiabetic and nonlymphopenic inbred diabetes-resistant (DR) BB rat line. Diabetes segregated as a single, autosomal recessive trait and was always accompanied by lymphopenia. Among the limited number of differences in the genomic DNA sequences of the two lines, DP and DR BB, one may account for the development of diabetes and lymphopenia in the DP BB rats. It may be possible to screen the genomic DNA for such differences to detect a marker for the phenotypes.

Antigen-dependent immunotherapy of non-obese diabetic mice with immature dendritic cells.

Immunotherapy can be used to induce immunological tolerance by a number of different protocols. During the last decade the ability to use tolerogenic dendritic cells (DCs) to prevent autoimmunity has received much attention. Many studies have attempted to use immature or semi-mature DCs to induce tolerance in the non-obese diabetic (NOD) mouse model of human type 1 diabetes. However, most studies to date have used protocols in which generation of DCs involved a culture step in fetal bovine serum (FBS)-supplemented medium which may affect tolerance induction in a non-specific fashion. Indeed, several studies have shown that DCs cultured in the presence of FBS will induce a powerful T helper type 2 (Th2) immune response towards FBS-related antigens which can suppress an ongoing immune response. Hence, this may interfere with diabetes development in the NOD mouse by induction of immune deviation rather than by antigen-specific tolerance. In order to test whether antigen-specific tolerance induction by DC therapy is feasible in the NOD mouse, we therefore generated immature DCs using autologous serum [normal mouse serum (NMS)-supplemented cultures] instead of FBS, and we show that these DCs can protect NOD mice from diabetes, if pulsed with insulin-peptide antigens before adoptive transfer. Our data therefore support that DC therapy is able to prevent diabetes in the NOD mouse in an antigen-specific manner.

Lack of systematically found insulin autoantibodies in spontaneously diabetic BB rats.

Insulin autoantibodies (IAAs) occur in newly diagnosed human insulin-dependent diabetes mellitus (IDDM) patients, but their presence in BB rats is controversial, possibly due to assay differences or variability in the animals studied. To resolve this controversy, IAAs were measured in well-characterized inbred BB rats both in radioligand assays with 125I-labeled rat insulin I or II, respectively, and in an enzyme-linked immunosorbent assay (ELISA) with rat insulin as antigen. In prospective studies, a total of 57 serums from 16 diabetes-prone (DP) BB rats were obtained during an interval ranging from 15 wk to the last week before onset and at onset of diabetes. At comparable ages, 21 serums were obtained from 8 DP BB rats not developing diabetes, and 70 matched serums were obtained from 19 diabetes-resistant (DR) BB rats. Levels of antibody binding increased slightly with increasing age in DP and matched DR rats. Two rats were positive at onset of IDDM in all assays but not in earlier samples. Otherwise, only few isolated serums from both types of rats regardless of diabetes had increased binding in one of the assays. In a cross-sectional study, the insulin-binding levels in 150-day-old DP rats (n = 20) that had not yet developed diabetes did not correlate with insulitis present in 3 of 20 rats and did not differ from 150-day-old DR BB rats (n = 20).(ABSTRACT TRUNCATED AT 250 WORDS)

Neonatal tolerization with glutamic acid decarboxylase but not with bovine serum albumin delays the onset of diabetes in NOD mice.

To test the role of glutamic acid decarboxylase (GAD65) or bovine serum albumin (BSA) autoimmunity in the pathogenesis of diabetes, GAD65 or BSA was injected intraperitoneally into neonatal female NOD mice (100 micrograms/mouse of each protein). Treatment with GAD65, but not with BSA, significantly delayed the onset of diabetes compared with control mice (P < 0.05). At 18 weeks, 6 of 10 control mice compared with 0 of 10 GAD65-treated mice (P = 0.005) and 7 of 14 BSA-treated mice had developed diabetes. However, after 79 weeks, 6 of 10 of the GAD65-treated mice were diabetic compared with 9 of 10 of the control mice and 12 of 14 of the BSA-treated mice. In GAD65-treated mice without diabetes, insulitis was markedly reduced compared with control or BSA-treated mice (P < 10(-4)). To further elucidate why GAD becomes an autoantigen, the expression in NOD mice islets was studied. Quantitative immunohistochemistry revealed that islet cell expression of GAD was increased in 5-week-old NOD mice compared with BALB/c mice (P = 0.02). With the occurrence of insulitis (9-15 weeks), the GAD expression was further increased relative to 5-week-old NOD mice (P < 0.02). In conclusion, GAD, but not BSA, autoimmunity is important for the development of diabetes in NOD mice. Furthermore, concordant with the appearance of insulitis, the GAD expression increased in NOD mouse islets, which could possibly potentiate the beta-cell-directed autoimmunity.

Derivation of non-lymphopenic BB rats with an intercross breeding.

Previous studies have suggested that the development of diabetes in the BB rats does not require the expression of T lymphopenia. In order to derive non-lymphopenic diabetic rats and define the relationship between the T cell abnormalities, MHC genotype, and diabetes, we performed a cross between BB/H and diabetes resistant BB/control followed by an intercross of the F1. In the F2, the overall incidence of diabetes and lymphopenia was 30% and 27%, respectively. Lymphopenia was strongly associated with diabetes (p less than 0.001) and was seen in 76% of the diabetic F2's. However, 6 of the diabetic were non-lymphopenic (24%) and 3 of the non-diabetics were lymphopenic (5%). In the non-lymphopenic diabetic animals, all T cell levels were within the normal range, but diabetes occurred at an earlier age than their lymphopenic littermates (p less than 0.001). In contrast to the strong association between the inheritance of lymphopenia and diabetes, no relationship between diabetes and Class I MHC restriction fragment length polymorphisms was found. We conclude: 1) Diabetes and lymphopenia are strongly associated inherited abnormalities in the BB rat and are not associated with Class I RFLP defined genotypes within the RTIu haplotype, 2) Animals in whom diabetes occurs in the absence of lymphopenia can be derived using this breeding approach 3) In our non-lymphopenic rats, diabetes occurred at an earlier age possibly reflecting the restoration of quantitative or qualitative T cell defects found in lymphopenic BB rats.

Interleukin-1 beta (IL-1) does not reduce the diabetes incidence in diabetes-prone BB rats.

The cytokine interleukin 1 beta (IL-1) has been implicated as a pathogenetic factor in the initial events leading to insulin-dependent diabetes mellitus. Previous studies investigating the impact of IL-1 on diabetes incidence in spontaneously diabetic rodent models have been conflicting. IL-1 induces anorexia and previous studies are hampered by the lack of pair-fed controls to the IL-1 treated animals. We report that daily injections of 4.0 micrograms/kg/day of recombinant human IL-1 (rhIL-1) for 13 weeks from 25-30 days of age did not alter the incidence of diabetes in the diabetes-prone (DP) BB rats (75%) when compared to pair-fed, vehicle treated controls (55%, p = 0.18), or to unhandled DP BB rats (80%, p = 0.71). However, IL-1 induced significantly higher blood glucose concentrations in the prediabetic period (p < 0.00005) and at diabetes onset (p < 0.00005) in the DP BB rats and caused episodes of blood glucose concentrations > 11 mmol/l in the prediabetic period in 11/20 DP BB rats compared to 4/27 diabetes-resistant (DR) BB rats and 4/28 Wistar Furth (WF) rats (both p < 0.004), compared to DP BB). Further, rhIL-1 induced fever in 11 weeks in the DP BB rats compared to 3 weeks in the DR BB and 6 weeks in the WF rats. Using high performance size exclusion chromatography specific anti-rhIL-1-antibodies were demonstrated in DR BB and WF, but not in DP BB rats. These antibodies neutralized the inhibitory effect of rhIL-1 on insulin secretion from isolated islets of Langerhans in vitro. The reduced pyrogenic and endocrine effect of rhIL-1 in the DR BB and WF rats compared to the DP BB rats could be explained by the impaired ability of the DP BB rats to produce anti-rhIL-1-antibodies. In conclusion, administration of rhIL-1 modulated the prediabetic period, and produced higher blood glucose concentrations at diagnosis, but did not change the diabetes incidence in DP BB rats. The results are not in conflict with the hypothesis that IL-1 is a pathogenetic factor in IDDM, caused by high local concentrations of rat IL-1 in the islets during early insulitis. The results also show the necessity of pair-feeding of the control group to the rhIL-1 group when interpreting data from experiments investigating rhIL-1 effects on diabetes development in animal models.

Reduced pancreatic insulin is associated with retarded growth of the pancreas in young prediabetic BB rats.

The beta-cell function in the perfused pancreas, the total pancreatic insulin content, and the weights of pancreas, kidney, spleen, and total body fat were compared at the age of 30 and 45 days in diabetes-prone and body weight-matched diabetes-resistant male BB rats. These inbred rats had an incidence rate of diabetes at 90 days of age of 80 and 0%, respectively. At day 30, the pancreatic insulin content was reduced in the diabetes-prone (n = 10) rats to 15 micrograms (range 7.7-31.8) compared with 32 micrograms (range 23.3-52.9) in the diabetes-resistant (n = 10) rats (p less than 0.01). Although the kinetics of insulin release in response to glucose was maintained, the amount of insulin released in the diabetes-prone rats was decreased (p less than 0.05) including the first peak (p less than 0.05). The weight of the spleen was less (p less than 0.05); whereas the weights of the pancreas, kidney, and total body fat did not differ from those observed in the diabetes-resistant control rats. At 45 days of age, the weight of the pancreas in the diabetes-prone rats (n = 8) was reduced to 159 mg (range 100-190) from 202 mg (range 185-214) (p less than 0.01). The body weight and body fat were identical to those observed in the diabetes-resistant controls (n = 8) and their kidney (p less than 0.05) and spleen (p less than 0.01) were larger. The insulin content was reduced to the same magnitude as the reduction of pancreatic weight.

Decreased levels of serum alpha-isoamylase prior to diabetes onset in BB rats.

The development of insulin-dependent diabetes mellitus (IDDM) includes a prodrome of autoimmunity against pancreatic beta cells. The period of subclinical islet cell disease with altered beta-cell function may be prolonged. We have determined the serum pancreatic alpha-isoamylase in both young diabetes-prone (DP) and newly diabetic BB rats to test whether changes in the pancreas prior to IDDM are reflected by this enzyme, shown to be regulated by insulin. A prospective analysis of inbred BB rats (n = 28) that later developed diabetes showed that the alpha-isoamylase at the time of onset was reduced by 19% (p less than 0.02) compared with levels observed 1 week earlier and by 30% (p less than 0.01) compared with levels 2 weeks before onset. Furthermore, when compared to age-matched diabetes-resistant (DR) BB rats in a cross-sectional study, the DP BB rats investigated in groups at 20, 30, 40, 50, 60, and 70 days of age had significantly lower (p less than 0.01) serum alpha-isoamylase already from 50 days of age, which is 2-6 weeks prior to the expected onset of diabetes. Finally, in 70-day-old cofostered DP and DR male rats with identical body weight and rates of growth, the serum alpha-isoamylase was decreased in the DP yet nondiabetic (n = 8) rats compared with the DR (n = 8) rats (p less than 0.05). Reduced levels of serum alpha-isoamylase, therefore, may reflect loss of beta cells or beta-cell function in the pancreas of diabetes-prone but not yet diabetic BB rats.

Insulitis and diabetes are preceded by a decrease in beta cell volume in diabetes-prone BB rats.

Immunocytochemistry combined with morphometry was used to test the hypothesis that insulitis and diabetes are preceded by quantitative changes in the pancreas of diabetes-prone BB (DPBB) rats. Diabetes-resistant BB (DRBB) rats of the w-subline served as controls. In the first part of the study rats aged 15, 30, 45, and 60 days were studied. At 60 days preceding both insulitis and onset of diabetes, the DPBB rats demonstrated lower volumes of the entire pancreas, the parenchyma, the endocrine pancreas, and beta, A, and D cells. Body weights of the DPBB rats were lower than the DRBB rats from 15 days of age on. In the second part of the study, DP and DRBB rats were coreared by a foster mother to obtain weight-matched animals. Morphometric analysis at 70 days of age revealed a reduction in both the beta cell volume density and volume whereas no differences were seen in other pancreatic parameters. These results indicate that the appearance of insulitis and the later onset of insulin-dependent diabetes are preceded by a reduction in beta cell volume.

Leukocytosis at the onset of diabetes in crosses of inbred BB rats.

Inbred lymphopenic, diabetes-prone (DP) and non-lymphopenic, diabetes-resistant (DR) BB rats in a specific pathogen-free (SPF) colony were subjected to a cross-intercross breeding experiment which showed diabetes to segregate as a recessive trait. All DP rats, but none of the DR and F1 rats, developed diabetes. In contrast, about 25% of the F2 rats developed diabetes which made it possible to study these rats without maternal influence of diabetes. All rats were bled at regular intervals between 30 and 150 days of age, and the samples analyzed for numbers of leukocytes, lymphocytes, neutrophils, monocytes and eosinophils. Leukocyte numbers tended to increase with age until about 100 days, and to decline thereafter. Males had more leukocytes than females. Coinciding with the time of onset of overt diabetes, there was a large increase in eosinophils, along with smaller increases in neutrophils, monocytes and lymphocytes. These data in SPF DP and DR BB rats and their cross-intercross offspring demonstrate that the overt onset of diabetes is associated with a significant leukocytosis.

Decreased weight gain in BB rats before the clinical onset of insulin-dependent diabetes.

Inbred specific pathogen-free diabetes-prone (DP) and diabetes-resistant (DR) BB rats were crossed to produce F1 and intercrossed to produce F2 rats. Diabetes segregates in these crosses as a recessive trait on rat chromosome 4. The weight gain of genetically diabetes-prone rats born to F1 healthy parents was studied to avoid effects of maternal diabetes. The weight gain of the F2 rats was initially not different from the F1 parents. The F2 rats later developing diabetes grew in parallel with their non-affected siblings up until the last 9 days before onset. During these 9 days they showed a decreased weight gain compared to their healthy litter-mates regardless of age. We conclude that decreased weight gain precedes the abrupt clinical onset of diabetes in BB rats and that it may be due to processes associated with the selective loss of beta cells.

Characteristics of rat CD4(+)CD25(+) T cells and their ability to prevent not only diabetes but also insulitis in an adoptive transfer model in BB rats.

Human and mouse CD4(+)CD25(+) T cells have been intensively studied through the last decade. However, little is known about this subset in other species. This study describes the phenotype of rat CD4(+)CD25(+) Foxp3(+) T cells and the site in which they exert regulation in a transfer-induced autoimmune diabetes model. Several proteins and mRNAs are up-regulated in unstimulated rat CD4(+)CD25(+) T cells compared with CD4(+)CD25(-) T cells, including Foxp3, Lag-3, CD80, interleukin 10 (IL-10) and CTLA-4. To investigate CD4(+)CD25(+) T cells in vivo, we transferred three million diabetogenic T cells either alone or in combination with two million CD4(+)CD25(+) T cells to 30-day-old BB rats. The pancreas and the pancreatic lymph nodes were examined as two potential regulatory sites. Time-course analysis of pancreatic histology following diabetogenic T-cell transfers revealed insulitis from about 14 days after transfer. By contrast, rats receiving both diabetogenic T cells and CD4(+)CD25(+) T cells had no insulitis at any time. Moreover, the frequency of diabetogenic T cells in the pancreatic lymph nodes 2 days after transfer was significantly reduced in rats receiving both subsets. These data indicate that the primary site of T-cell regulation is in the draining lymph nodes and not the pancreas in our model.

Comparative mapping of the human homologue of the rat diabetes susceptibility gene lyp to a 1.3-Mb segment on HSA7.

The rat diabetes susceptibility gene, Lyp or Lymphopenia, has been localized to RNO4. Proximal to Lyp are the genes caspase-2 (Casp2) and pancreatic trypsin 1 (Prss1), while neuropeptide Y (Npy) is the closest distally positioned gene. In human, the three genes are syntenic on HSA7, but they are not on a conserved segment: CASP2 and PRSS1 are localized to 7q35, while NPY is localized to 7p15.1. This raises the question whether the human homologue of Lyp is linked to CASP2/PRSS1 or to NPY. We present a comparative map of the Lyp region in rat and human, assigning the gene to a 1.3-Mb segment between RNY3 and ABP1 at 7q35.

A sequence-ready PAC contig of a 550-kb region on rat chromosome 4 including the diabetes susceptibility gene Lyp.

The Lyp locus controls diabetes development in rats. The diabetogenic allele in diabetes-prone BB rats is responsible for T cell lymphopenia characterized by the absence of regulatory T cells. We present refined genetic and radiation hybrid maps of the Lyp region on rat chromosome 4, a single 800-kb rat yeast artificial chromosome and a rat P1-derived artificial chromosome (PAC) contig corresponding to approximately 550 kb, both encompassing the entire candidate region. The contig, consisting of 48 PACs, gives 3- to 12-fold coverage. Genetic, radiation hybrid, and physical data were all in agreement and supported the same marker order. Nine genes and ESTs were identified in the contig in addition to a rat EST from the University of Iowa rat EST database-all possible candidate genes for Lyp. Alignment of our rat PAC contig with sequenced human PAC/BAC contigs confirms the position within the region of 3 of the 10 candidates and identifies an additional 8 genes/ESTs as candidates. These data will facilitate identification of Lyp.

Segregation of autoimmune type 1 diabetes in a cross between diabetic BB and brown Norway rats.

Diabetes-prone DP-BB rats spontaneously develop insulin-dependent diabetes mellitus resembling type 1 diabetes mellitus in man. Expression of T cell lymphopenia and presence of at least one class II major histocompatibility complex (MHC) RT1u haplotype are required for development of diabetes. Diabetes segregation was studied in lymphopenic backcross (BC) offspring from a cross between male DP-BB/HRI and female BN/Mol rats. Diabetes occurred in 75% of BC rats with genotype RT1u/u and in 18% of those being RT1n/u in genotype. The latter developed diabetes significantly later than MHC homozygotes and parental DP-BBs. Our data further point to the existence of additional genes of minor importance for development of IDDM. One of these seemed to be positioned on the X chromosome. The recently published linkage to chromosome 18 could not be confirmed however. Finally, the BN-derived non-albino allele of the C gene was associated with higher diabetes incidence. This points to the existence of minor susceptibility genes in other strains of rats.

Mapping of caspase-2 in the rat and its exclusion as a candidate gene for lymphopenia.

Caspase-2 is a member of the caspase family of cystein proteases involved in programmed cell death or apoptosis. Functional and genetic data suggest it as a candidate gene for lymphopenia (Lyp)-a susceptibility gene for rat diabetes-which is responsible for the T-cell lymphopenia in the diabetes-prone BB rat. Firstly, there is a higher frequency of apoptosis among recent thymic emigrants in the diabetes-prone BB rat than in the non-lymphopenic diabetes-resistant BB rat. Secondly, caspase-2 maps close to Tcrb on mouse Chromosome (Chr) 6, while Lyp is closely linked to Tcrb on the homologous rat Chr 4. In this paper, we report genetic fine-positioning and radiation hybrid mapping of caspase-2 in the rat. Both methods positioned caspase-2 to rat Chr 4 between markers Prss1 and D4Mit5. Since Lyp maps distally to D4Mit5, between markers D4Rat75 and Npy, we exclude caspase-2 as a candidate gene for Lyp.

A pronounced thymic B cell deficiency in the spontaneously diabetic BB rat.

In an attempt to elucidate the origin of the T cell lymphopenia and/or the beta-cell-specific autoimmunity observed in diabetes-prone Bio-Breeding (DP-BB) rats, a thymic cDNA library was subjected to differential screening with thymic cDNA probes of DP-BB rats and nonlymphopenic nondiabetic controls. This approach resulted in the identification of a prominent lack of thymic B cells in DP-BB rats. This deficiency is distinct from a less pronounced peripheral B cell deficiency of different timing. The thymic B cell defect is linked to the lymphopenia trait on chromosome 4 and thereby with susceptibility to diabetes in crosses involving the DP-BB rat. In conclusion, our data suggest that the contribution of thymic B cells to the (negative) selection of thymocytes is inadequate in DP-BB rats, thus providing a plausible explanation for at least some of the spontaneous autoimmune phenomena in this animal model.

Transplantation of purified islet cells in diabetic BB rats.

The ability to prepare purified islet Beta-cell aggregates was used to examine the survival of this cell type after allotransplantation in diabetic BB rats. The aggregates were intraportally implanted in numbers that were previously found to correct a streptozotocin-induced diabetic state in syngeneic or allogeneic Brown Norway recipients. When the grafts were prepared from RT1u/l donors, which shared the MHC-class I antigen with the BB recipients (RT1u/u), their implant sites became diffusely infiltrated by inflammatory cells and their metabolic function was completely lost within 5 weeks. MHC-class I incompatible islet Beta-cell allografts (RT1n/n) exhibited a longer survival, in particular when combined with other islet endocrine cells and/or when covered by a 5-week cyclosporin treatment. In the latter combination, 10 of 12 BB rat recipients remained normoglycaemic over the 10-week observation period, their liver implants presenting a comparable insulin reserve and similarly discrete mononuclear cell infiltration as streptozotocin-diabetic Brown Norway rats receiving this treatment. However, administration of cyclosporin to diabetic BB rats was associated with a morbidity that was not observed in drug-treated streptozotocin-diabetic Brown Norway animals or in untreated diabetic BB rats. It is concluded that MHC-incompatible islet Beta cells can induce a long-term normalization in diabetic BB rats provided that they are implanted under conditions which allow allograft acceptance. The standardized preparation of purified islet Beta-cell grafts can help assessing the conditions for successful transplantations in diabetes with an autoimmune origin.