BioBreeding Research Articles

GAD65 and Insulin B Chain Peptide (9-23) are not Primary Autoantigens in the Type 1 Diabetes Syndrome of the BB Rat

To investigate whether GAD65 whole molecule, GAD65 p35 or insulin B chain peptide (amino acids 9-23) play an essential role in the pathogenesis of type 1 diabetes in the BioBreeding (BB) rat, we gave serial injections of GAD65, p35 or insulin B chain (9-23) to six groups of BB/Worcester rats. The individual antigens were administered either intrathymically on day 2 and intraperitoneal in MF 59-0 adjuvant 5 times during the first 5 weeks, or by intranasal instillation once neonatally and 5 days/week for the following 6 weeks. Control groups were injected with vehicle only. Age of onset of diabetes and degree of insulitis were not different between controls and antigen-treated rats. Rats that received GAD65 intrathymically and intraperitoneal!) developed high GAD65-antibody titers without altering diabetes development. In GAD65-treated animals, serum antibodies recognized epitopes at 3 sites on GAD65 in diabetic animals but only at 1 site in non-diabetic animals. GAD65-injected animals also showed a significant reduction of IFN-γ mRNA expression in the thymus. This study provides evidence against the hypothesis that GAD65 and insulin B chain peptide (9-23) are primary diabetogenic autoantigens in BB rats because immunizations with these antigens and GAD65-induced immune deviation did not alter the development of diabetes.

Significant role of intragraft lymphoid tissues in preventing insulin-dependent diabetes mellitus recurrence in whole pancreaticoduodenal transplantation.

Graft recurrence of insulin-dependent diabetes mellitus (IDDM) was examined. Islet transplantation or pancreas-alone transplantation excluding the duodenum and peripancreatic lymph nodes was compared with whole pancreaticoduodenal transplantation. A Wistar Furth (WF; RT1(u), RT6.2) to major histocompatibility complex (MHC)-compatible diabetes-prone (DP; RT1(u), RT6.1 gene carrier)-biobreeding (BB) rat transplantation model was used. Only DP recipients that had been transplanted with whole pancreaticoduodenal grafts were free from IDDM recurrence (>60 days postgrafting) when treated with anti-intercellular adhesion moluecule-1 (ICAM)-1/leukocyte function-associated antigen-1 (LFA-1) monoclonal antibodies (mAbs). In the spleen cells of the DP rats that had accepted pancreatic grafts (60 days postgrafting), flow cytometric analysis showed that NKR-P1(+)TCRalphabeta(+) (NKT) cells had proliferated markedly, with the proportion of 12.8 +/- 1.7% in the total splenic T cells, most of which (86.2%) were derived from the donor (RT6.2(+)). By enzyme-linked immunonosorbent assay (ELISA), serum interferon gamma (IFN-gamma) was not detected (<13 pg/ml) in all rats. However, interleukin-4 (IL-4) was detected as 158.8 +/- 28.0 pg/ml in the nonrecurrent DP recipients. These data suggested that to prevent IDDM recurrence in the pancreatic graft, the lymphocytes in the pancreaticoduodenal grafts are necessary. Also, the donor-derived NKT cells might have some immunoregulatory functions with a Th2 deviation.

Significant role of intragraft lymphoid tissues in preventing insulin‐dependent diabetes mellitus recurrence in whole pancreaticoduodenal transplantation

Graft recurrence of insulin-dependent diabetes mellitus (IDDM) was examined. Islet transplantation or pancreas-alone transplantation excluding the duodenum and peripancreatic lymph nodes was compared with whole pancreaticoduodenal transplantation. A Wistar Furth (WF; RT1u, RT6.2) to major histocompatibility complex (MHC)-compatible diabetes-prone (DP; RT1u, RT6.1 gene carrier)-biobreeding (BB) rat transplantation model was used. Only DP recipients that had been transplanted with whole pancreaticoduodenal grafts were free from IDDM recurrence (>60 days postgrafting) when treated with anti-intercellular adhesion moluecule-1 (ICAM)-1/leukocyte function-associated antigen-1 (LFA-1) monoclonal antibodies (mAbs). In the spleen cells of the DP rats that had accepted pancreatic grafts (60 days postgrafting), flow cytometric analysis showed that NKR-P1+TCRαβ+ (NKT) cells had proliferated markedly, with the proportion of 12.8 ± 1.7% in the total splenic T cells, most of which (86.2%) were derived from the donor (RT6.2+). By enzyme-linked immunonosorbent assay (ELISA), serum interferon gamma (IFN-γ) was not detected (<13 pg/ml) in all rats. However, interleukin-4 (IL-4) was detected as 158.8 ± 28.0 pg/ml in the nonrecurrent DP recipients. These data suggested that to prevent IDDM recurrence in the pancreatic graft, the lymphocytes in the pancreaticoduodenal grafts are necessary. Also, the donor-derived NKT cells might have some immunoregulatory functions with a Th2 deviation. © 1999 Wiley-Liss, Inc. MICROSURGERY 19:338–343 1999

Effect of long-term treatment with a purified micronized flavonoid fraction on pancreatic mononuclear cell infiltration in diabetic BB rats.

Bio Breeding (BB) rats develop a genetically determined insulin-dependent diabetes, because of the early destruction of pancreatic beta cells of Langerhans islets, massively infiltrated by inflammatory mononuclear cells. S 5682, registered as Daflon, 500 mg, is a purified micronized flavonoid fraction (90% diosmin, 10% hesperidin), which has been shown to possess antiinflammatory properties, including anti-free radical activity, effects on vascular permeability, venous tone, and perivenous inflammation. We studied the effect of S 5682 on the course of pancreatic insulitis in diabetic BB rats. All the diabetic BB rats were hyperglycemic, with an increase of plasma levels of fructosamine, alpha-1 acid glycoprotein, and fibrinogen, and a dramatic decrease of C-peptide level. These parameters were not modified by S 5682. Pancreas histologic studies showed that in S 5682-treated diabetic BB rats, lymphocytic infiltration of Langerhans islets was less important and frequent than in untreated diabetic BB rats. By quantitative analysis, a highly significant difference was observed for insulitis, as well as perivasculitis, between S 5682-treated and untreated diabetic BB rats. This inhibitory effect of S 5682 on pancreatic mononuclear cell infiltration may be useful for a complementary treatment to decrease the development of insulitis in human insulin-dependent diabetes mellitus.

High Frequency Apoptosis of Recent Thymic Emigrants in the Liver of Lymphopenic Diabetes-Prone BioBreeding Rats

Diabetes-prone (DP) BioBreeding (BB) rats develop spontaneous autoimmune diabetes. DP-BB thymocyte export is reduced, and most thymic emigrants disappear rapidly from peripheral lymphoid tissues. DP-BB rats are consequently lymphopenic and circulate severely reduced numbers of T cells. Peripheral T cells present are phenotypically immature (Thy1+) and appear activated. We hypothesized that DP-BB recent thymic emigrants have a shortened life span and disappear by apoptosis. The percentage of T cells with an alphabetaTCR(low) B220+ CD4- CD8- phenotype was increased in DP peripheral lymphoid tissues when compared with normal, nonlymphopenic diabetes-resistant (DR) BB rat tissues. There was no evidence of DNA fragmentation in freshly isolated DP- or DR-BB rat cells, but, after 24 h of culture, a higher proportion of DP- than DR-BB splenic T cells underwent apoptosis. We then tested the hypothesis that BB rat T cells with the alphabetaTCR(low) B220+ CD4- CD8- phenotype accumulate and undergo apoptosis in the liver. Such cells were observed undergoing apoptosis in both DP- and DR-BB rats, but comprised approximately 80% of intrahepatic T cells in DP vs approximately 20% in DR-BB rats. Most alphabetaTCR(low) B220+ CD4- CD8- cells in the liver were also Thy1+. The data suggest that T cell apoptosis in the DP-BB rat is underway in peripheral lymphoid tissues and is completed in the liver. Increased intrahepatic apoptosis of recent thymic emigrants appears in part responsible for lymphopenia in DP-BB rats and the concomitant predisposition of these animals to autoimmunity.

Lack of recurrence of insulin-dependent diabetes mellitus in syngeneic and allogeneic islet-transplanted diabetic biobreeding rats.

Previous reports on experimental islet transplantation in animal models of human insulin-dependent diabetes mellitus show that islet grafts are susceptible to autoimmune destruction similar to that seen in native pancreatic islets. In this study, we demonstrated a lack of disease recurrence in diabetic BioBreeding (BB) rats after syngeneic and allogeneic islet transplantation. Four hundred to 1200 islets from BB (RT1u) and Lewis (RT1(1)) donors, isolated with stationary collagenase digestion and Ficoll density purification, were intraportally transplanted into spontaneously diabetic BB rats. The recipients received no immunologic manipulations before or after islet transplantation. When more than 900 syngeneic islets or when more than 600 allogeneic islets were transplanted, BB recipients remained normoglycemic for over 280 days, irrespective of age at onset, duration of exogenous insulin treatment, or age at transplantation. When at least 500 islets were transplanted, the recipients survived for a long period with normoglycemia or in a noninsulin-dependent diabetic state. Upon histological examination, mononuclear cell infiltration was observed in every islet graft examined, but the severity of infiltration in most of the grafts was mild to moderate. These results indicate that the islet grafts in the BB recipients were destroyed extremely slowly. It is conceivable that in our BB colony, a state of immunologically low responsiveness that allows diabetic animals to accept syngeneic or allogeneic islet grafts, occurs around the onset period and becomes more pronounced with aging. Our BB rat colony can be considered to be a novel substrain with unique immunological characteristics.

Genetic isolation of iddm 1 on chromosome 4 in the biobreeding (BB) rat.

Genetic isolation of iddm 1 on chromosome 4 in the biobreeding (BB) rat.

Cellular and Molecular Mechanisms for the Initiation and Progression of β Cell Destruction Resulting from the Collaboration Between Macrophages and T Cells

Insulin-dependent diabetes mellitus (IDDM) is caused by the progressive autoimmune destruction of insulin-producing pancreatic β cells. Although the pathogenesis of autoimmune IDDM has been extensively studied, the precise mechanisms involved in the initiation and progression of β cell destruction remain unclear. Animal models used in the study of IDDM, such as the BioBreeding (BB) rat and the nonobese diabetic (NOD) mouse, have greatly enhanced our understanding of the pathogenic mechanisms involved in this disease. In these animals, macrophages and/or dendritic cells are the first cell types to infiltrate the pancreatic islets. Macrophages must be involved in the pathogenesis of IDDM early on, since inactivation of macrophages results in the near-complete prevention of insulitis and diabetes in both NOD mice and BB rats. The presentation of β cell-specific autoantigens by macrophages and/or dendritic cells to CD4+ T helper cells, in association with MHC class II molecules, is considered the initial step in the development of autoimmune IDDM. The activated macrophages secrete IL-12, which stimulates Thl type CD4+ T cells. The CD4+ T cells secrete IFN-γ and IL-2. IFN-γ activates other resting macrophages, which, in turn, release cytokines, such as 1L-1 β, TNF-α, and free radicals, which are toxic to β cells. During this process, IL-2 and other cytokines induce the migration of CD8+ peripheral T cells to the inflamed islets, perhaps by inducing the expression of a specific homing receptor. The precytotoxic CD8+ T cells that bear β cell-specific autoantigen receptors differentiate into cytotoxic effector T cells upon recognition of the β cell-specific peptide bound to MHC class I molecules in the presence of β cell-specific CD4+ T helper cells. The cytotoxic CD8+ T cells then effect β cell damage by releasing perforin and granzyme, and by Fas-mediated apoptosis. In this way, macrophages, CD4+ T cells, and CD8+ T cells synergistically destroy β cells, resulting in the onset of autoimmune IDDM.

Phenotypic characterization of mononuclear inflammatory cells in salivary glands of bio-breeding rats

The purpose of this study was to assess whether mononuclear cell abnormalities exist in salivary glands from autoimmune Bio-Breeding (BB) rats. Frozen sections of gland tissues were prepared from five diabetes-resistant BB rats (BB-DR), from five BB rats with diabetes (BB-DP) and from five Wistar rats. A panel of six monoclonal antibodies was used to identify membrane antigens associated primarily with monocytes (ED1), mature tissue macrophages (ED2), lymphoid macrophages (ED3), MHC class II (Ia) antigen (0X6), CD5+ T lymphocytes (0X19), and rat B lymphocytes (0X33). Normal submandibular, sublingual and parotid glands contained few EDI-positive cells, usually two or fewer per field. Tissue macrophages identified by clone ED2 comprised a major mononuclear cell subset in both Wistar and BB rats. However, the number of ED2-positive mononuclear cells was significantly depressed in the submandibular and parotid glands from BB-DR and BB-DP animals, being present in quantities 25–50% of those observed in glands from normal Wistar rats ( p < 0.001). In contrast, 25- to 30-fold greater numbers of ED3-positive macrophages were observed in submandibular glands from BB rats ( p < 0.001). MHC class II (Ia) antigen expression also was 4− to 6-fold greater in BB rat submandibular glands, compared to Wistar rats ( p < 0.001). CD5+ T-lymphocytes were rare or entirely absent in BB sublingual glands (0 to 1 cell per 0.87 mm 2 field), compared to 47 cells per field from Wistar sublingual glands. No B lymphocytes were identified with antibody OX33 in any of the rat strains. These findings indicate that BB rat salivary glands differ significantly from Wistar salivary glands. In BB rats there is a rich population of ED3-positive macrophages and T lymphocytes in submandibular gland, low quantities of T lymphocytes in sublingual gland, and fewer ED2-positive macrophages in all three major salivary glands. These differences in mononuclear cell subpopulations may also influence salivary gland function in mucosal immunity.

Kidneys from normotensive donors lower blood pressure in young transplanted spontaneously hypertensive rats.

Single transplanted kidneys from spontaneously hypertensive rats (SHR) have been shown to elicit hypertension in genetically normotensive recipients. This study was designed to investigate the effects of single transplanted kidneys from genetically normotensive donors [Biobreeding (BB)/Ottawa Karlsburg (OK) rats] on blood pressure in SHR recipients. The following groups were formed: group 1 (n = 11), SHR donors and SHR recipients; group 2 (n = 15), BB/OK donors and SHR recipients; and group 3 (n = 8), BB/OK donors and BB/OK recipients. Recipients received antihypertensive treatment (hydralazine) from weaning until renal transplantation at the age of 9 wk and immunosuppressive treatment (anti-CD4 antibody and cyclosporine A) for 3 wk starting on the day of transplantation. Six weeks after transplantation, intra-arterially measured blood pressure and heart weight-to-body weight ratio were highest in group 1, intermediate in group 2, and lowest in group 3. There were no significant differences with respect to plasma urea and creatinine concentrations among the three groups. These results support the hypothesis that hypertension in renal-transplanted SHR depends in part on the genetic background of the transplanted kidney.

The lymphopenia (lyp) gene controls the intrathymic cytokine ratio in congenic BioBreeding rats.

The lymphopenia gene (lyp) on rat chromosome 4 is closely linked to autoimmune diabetes in the BioBreeding (BB) rat. Lyp controls the number of peripheral lymphocytes by reducing T cells of the RT6+ phenotype by almost 90%. Following nine cycle of marker-assisted cross-intercross breeding we have developed congenic lyp/lyp, lyp/+ and +/+ (wildtype) rats on the background of DR rats. Prediabetic and insulitis free lyp/lyp, lyp/+ and +/+ rats were used to determine the effect of lyp on cytokine expression in the thymus. In situ hybridization of thymus cryosections showed that the interferon gamma (IFN gamma) mRNA expression was highest in lyp/lyp rats and the hybridization signal was restricted to the medullary compartment. The frequency of IFN gamma and interleukin (IL)-10 mRNA expressing cells in isolated thymocytes determined by quantitative image analysis, demonstrated an increased IFN gamma: IL-10 ratio in thymocytes from lyp/lyp homozygotes compared to lyp/+ and +/+ rats. This confirmed a lyp gene dose-dependent segregation of the IFN gamma high phenotype. Recombinant human glutamic acid decarboxylase (GAD65) increased the number of IFN gamma and IL-10 mRNA expressing thymocytes after in vitro culture. We conclude that the quantitative ratio of cytokine producing thymocytes is associated with the lyp genotype. These potentially autoreactive thymocytes may explain the establishment of beta-cell directed autoimmunity in the BB rat despite peripheral lymphopenia.

Induction of insulin-dependent diabetes mellitus in PVG.RT1 u rats

THE BIOBREEDING (BB) rat strain is used to model human autoimmune insulin-dependent diabetes mellitus (IDDM). 1 The diabetes-resistant (DR) subline of the BB rat does not develop diabetes spontaneously, but is genetically predisposed to the disorder. 1 More than 90% of BB-DR rats become diabetic after combined treatment with a depleting monoclonal antibody (MAb) to remove RT6.1 + regulatory T cells and polyinosinic:polycytidylic acid (poly I:C), an inducer of interferons. 2 Lymph node T cells from diabetic BB-DR rats adoptively transfer IDDM to untreated histocompatible (RTI u) WAG- nu/nu rats. 3,4 Thymocytes from either normal or diabetic BB-DR rats also transfer IDDM provided that recipients are treated with anti-RT6.1 MAb to preclude maturation of RT6 + regulatory T cells from the transfused thymocytes. 5 We have previously observed that other rat strains that express the RT6.1 alloantigen, among them the YO (RTI u) and LEW (RT1 1) strains do not develop autoimmune diabetes when treated in the same way as BB-DR rats. 6 To investigate further if combination treatment with anti-RT6.1 MAb and poly I:C can induce autoimmunity in strains other than the BB, we studied major histocompatibility complex (MHC) congenic PVG.RTI u rats. These animals are phenotypically normal and free of spontaneous disease but reportedly develop IDDM after irradiation and thymectomy. 7

The BB rat as a model of human insulin-dependent diabetes mellitus.

Use of the BioBreeding (BB) rat to model human insulin-dependent diabetes mellitus (IDDM) is useful in that characteristics of diabetes in the BB rat closely parallel those observed in human IDDM. Diabetic animals can be biopsied, autopsied, and bred to study the genetic basis of IDDM. The genetic, immunological, and environmental components of the disease can all be investigated under controlled conditions. Two inbred lines of BB have been used in the majority of published studies using this model system; these rats are designated as diabetes prone (DP-BB/Wor) and diabetes resistant (DR-BB/Wor). This unit presents two protocols for the diagnosis and prevention of spontaneous IDDM in DP-BB/Wor rats. Two alternate protocols are given for the induction of autoimmune disease in DR-BB/Wor rats and for the adoptive transfer of autoimmunity into histocompatible athymic WAG nu/nu recipient rats. Support protocols are also given for diagnosis of insulitis, treatment of diabetic rats with insulin, and serological analysis of blood samples from sentinel rats to monitor the pathogen status of the colony.

Histomorphological aspects of the development of thyroid autoimmune diseases: consequences for our understanding of endocrine ophthalmopathy.

In this short review we will first evaluate the histomorphological aspects of the human and spontaneous animal thyroid autoimmune diseases. These diseases include Hashimoto goiter, primary myxedema, Graves' disease, and the spontaneous forms of thyroiditis in the Bio Breeding (BB) rat, the nonobese diabetic (NOD) mouse, and the obese strain (OS) of chicken. Based on sequential histomorphological events in the animal models of thyroid autoimmune disease, a mechanism for the pathogenesis of thyroid autoimmune disease is proposed. Since one of the human thyroid autoimmune diseases, specifically Graves' disease, is often associated with ophthalmopathy, the histomorphological aspects of the ophthalmopathic process are also evaluated to consider its possible autoimmune character.

Evidence that Th1 lymphocytes predominate in islet inflammation and thyroiditis in the BioBreeding (BB) rat.

Th1 cytokines are thought to play a key role in islet inflammation and destruction in insulin-dependent diabetes mellitus (IDDM). We studied this hypothesis in the diabetes-prone (DP)-BB and the diabetes-resistant (DR)-BB rats that are used as a model of human IDDM. The DP-BB rat develops spontaneous autoimmune diabetes at the age of 11-14 weeks. In the DR-BB rat, diabetes is inducible by depletion of RT6+ lymphocytes and coadministration of polyinosinic:polycytidylic acid (Poly I:C). We used reverse transcriptase-polymerase chain reaction (RT-PCR) and semi-quantitative PCR techniques to examine mRNA expression of Th1 and Th2 cytokines in inflamed islets and thyroids from DP-BB and DR-BB rats. We observed that in DP-BB and in treated DR-BB rats, the levels of TCR beta, IFN-gamma and IL-12p40 mRNA increase with disease progression. In contrast, expression of message for IL-2 and IL-4 is minimal to undetectable in DP-BB and RT6-depleted DR-BB animals at any age. Message for IL-10 is detectable in DP and DR islets; however, its level of expression does not change with disease progression. A similar cytokine mRNA profile is observed in inflamed thyroids from acutely diabetic RT6-depleted DR-BB rats. Incubation of 10 wk old DP islets for 48 h in the presence of anti-CD3 antibody, followed by an incubation with rIL-2 for an additional 5-7 days, results in an expansion of T lymphocytes, and these cells express high levels of IFN-gamma and IL-10 mRNA. Our results suggest that autoimmunity in DP-BB and DR-BB rats is mediated by Th1 lymphocytes and that IFN-gamma and IL-12 are likely to play a key role in islet and thyroid inflammation and destruction in IDDM.

Renal hypertrophy is associated with upregulation of TGF-beta 1 gene expression in diabetic BB rat and NOD mouse.

Renal hypertrophy is an early feature of diabetes, and it may predispose the kidney to the eventual development of parenchymal dysfunction. Since we have previously demonstrated that short-term culture in high glucose concentration stimulates production of transforming growth factor-beta 1 (TGF-beta 1) in proximal tubular and glomerular mesangial cells, we postulated that this cytokine, which has potent regulatory effects on cellular growth and extracellular matrix production, is important in mediating diabetic renal disease. In this study we evaluated the gene and protein expression of TGF-beta 1 in the kidney of two rodent models of spontaneous insulin-dependent diabetes mellitus [the biobreeding (BB) rat and the nonobese diabetic (NOD) mouse]. In association with the appearance in both models of significant renal hypertrophy, TGF-beta 1 mRNA levels were increased threefold in the kidney of the diabetic BB rat after 3 days of diabetes and also threefold after 7-9 days in the NOD mouse. There was no increase in TGF-beta 1 transcripts in the livers of the diabetic animals, suggesting that this response is tissue specific. Immunohistochemical studies revealed that TGF-beta 1 protein is concordantly elevated in the cortical tubular cells of the diabetic kidney in both models. These results suggest that the stimulated expression of renal TGF-beta is an early manifestation of the involvement of the kidney by diabetes. Whether increased TGF-beta production in the diabetic kidney is a key promoter of diabetic renal manifestations (e.g., hypertrophy) deserves additional studies.

Prevention of autoimmune type I diabetes in biobreeding (BB) rats by a newly established, autoreactive T cell line from acutely diabetic BB rats.

An autoreactive T cell line, BNY-2, was established from lymphocytes isolated from the islets of acutely diabetic BioBreeding (BB) rats after continuous stimulation of the isolated lymphocytes by irradiated lymph node cells. Flow cytometric analysis showed that BNY-2 cells were positive for CD4 and CD5 and were negative for CD8 and RT6, indicating that these cells were phenotypically Th cells rather than cytotoxic T cells. mAbs against rat MHC class II Ags (anti-RT1.D) blocked the proliferation response of BNY-2 cells, suggesting that these cells recognize the MHC class II Du molecule. When splenic T lymphocytes from diabetes-prone BB rats were stimulated by Con A in the presence of irradiated BNY-2 cells or irradiated lymph node cells, the BNY-2 cells had a significant suppressive effect on splenic T cell proliferation, whereas lymph node cells had no effect. When we injected diabetes-prone BB rats i.v. at 30 and 60 days of age with activated BNY-2 cells, the incidence of diabetes was significantly reduced compared with that seen in saline-injected control rats (diabetic incidences were 12 and 80%, respectively). On the basis of these observations, we conclude that autoreactive BNY-2 T cells, established from the pancreatic islets of acutely diabetic BB rats, can prevent the development of autoimmune diabetes in the diabetes-prone BB rat by an immunosuppressive effect.

Evidence that intrathymic islet transplantation does not prevent diabetes or subsequent islet graft destruction in RT6-depleted, diabetes-resistant BioBreeding/Worcester rats.

Pancreatic islet allografts transplanted intrathymically are accepted and restore normoglycemia in streptozotocin-diabetic rats given one injection of antilymphocyte serum. Intrathymic allografts similarly restore normoglycemia in diabetes-prone (DP) Bio-Breeding (BB) rats that have developed spontaneous autoimmune diabetes. Intrathymic islets also reduce the frequency of subsequent diabetes when transplanted prophylactically into young DP rats. These findings suggest that intrathymic transplantation can prevent not only allograft rejection, but also the appearance and recurrence of autoimmune tissue destruction. To explore these hypotheses further, we attempted both to confirm previous studies and to extend them to another model of autoimmune diabetes, the RT6-depleted diabetes-resistant (DR) BB rat. Fewer than 1% of DR-BB rats develop spontaneous diabetes, but most become hyperglycemic after in vivo immune elimination of RT6+ T cells. Using the protocols described in the literature, we observed the following: (1) Consistent with previous reports, intrathymic islet allografts survived indefinitely in streptozotocin-diabetic, antilymphocyte serum-treated, non-BB recipient rats. (2) Consistent with previous reports, intrathymic islet grafts produced long-term normoglycemia in diabetic DP-BB rats and also reduced the frequency of spontaneous diabetes in young animals transplanted prophylactically. (3) In contrast, intrathymic islets (iso- and allografts) neither prevented nor reversed diabetes in RT6-depleted DR rats. We hypothesize that intrathymic islet grafts survive in DP-BB rats because they are lymphopenic and immunocompromised, whereas immunocompetent diabetic DR rats successfully recapitulate the autoimmune disease process. Although intrathymic allograft transplantation is postulated to induce a state of tissue-specific tolerance, our results indicate that this tolerant state may not extend to autoimmune destruction of either isografts or allografts.

Adrenic acid Δ4 desaturation and fatty acid composition in liver microsomes of spontaneously diabetic Wistar BB rats

We examined the Δ4 (n-6) desaturation and the fatty acid composition of liver microsomes in the insulin-dependent spontaneously diabetic Wistar Bio-Breeding (BB) rat. The desaturation of adrenic acid to n-6 docosapentaenoic acid was decreased in the normo- and hyperglycemic diabetic rats. Insulin treatment with 1.0 IU. 100 g body weight −1 twice a day for 2 days restored the reduced activity during the hypoglycemic period. The pattern of responses was similar to that of linoleic acid Δ6 and dihomo-γ-linolenic acid Δ5 desaturases, with a non-parallel relationship between the desaturation system and the glycemia. The microsomal fatty acid composition of BB rat liver reflected only partially to the Δ4 desaturation at different states of glycemia. Factors other than impaired desaturation system are involved in the fatty acid metabolism of spontaneously diabetic rats.

The RT6 Rat Lymphocyte Alloantigen Circulates in Soluble Form

RT6 is a rat maturational lymphocyte alloantigen that appears to subserve important immunoregulatory functions. The lymphopenic diabetes-prone BioBreeding (BB)/Worcester rat is severely deficient in RT6+ T cells and develops spontaneous autoimmune diabetes mellitus. Transfusion of RT6+ T cells prevents the disease. Conversely, in vivo immune elimination of RT6+ T cells from the diabetes-resistant line of BB rats induces diabetes and thyroiditis. RT6 protein is expressed in two allotypic forms, each linked to the cell surface by a phosphatidylinositol (PI) anchor. The mechanism by which RT6+ T cells exert their regulatory function is not known, nor is the function of the RT6 protein defined. In this study, we investigated the possibility that, like other PI-linked proteins. RT6 also exists in a soluble from in the circulation. Using standard biochemical procedures we observed: (i) Soluble RT6 circulates in readily detectable amounts in all rat strains studied. (ii) The diabetes-prone BB rat circulates less RT6.1 than does any other strain, including the coisogenic diabetes-resistant line. (iii) Injections of monoclonal anti-RT6.1 antibody rapidly eliminate soluble RT6 from the circulation of diabetes resistant BB rats. The existence of a soluble form of a protein associated with immunoregulatory T cells suggests the possibility that soluble RT6 itself might possess immunomodulatory properties.