BioBreeding Rats Research Articles

The renal expression of transforming growth factor-beta isoforms and their receptors in acute and chronic experimental diabetes in rats.

Transforming growth factors-beta (TGF-beta) are fibrogenic factors that have been strongly implicated in the development of diabetic nephropathy. Our aim was to use two animal models [the streptozotocin (STZ)-induced diabetic rat and the genetically prone biobreeding (BB) rat] to fully characterize the responses of the renal TGF-beta system in both short- and long-term diabetes. In this study changes in the entire renal TGF-beta system, at both protein and messenger RNA (mRNA) levels, have been characterized using the techniques of immunocytochemistry, Western blotting, and ribonuclease protection assay. We also used Western blotting of pro-collagen-I C-peptide to demonstrate that the rate of fibrogenesis was highest over the first 2 weeks of diabetes. TGF-beta1, TGF-beta2, and receptor mRNA and protein were detected in the control nondiabetic kidney. It was found that dramatic and dynamic changes occur in all parts of the renal TGF-beta axis in both models of experimental diabetes, but TGF-beta2 and TGF-betaRII proteins were the predominant responsive element, particularly during the acute phase of disease. For example, during the acute phase of disease (0-30 days), although renal TGF-beta1 mRNA levels were elevated, no increases in the corresponding protein were detected in the kidney. By contrast, in the absence of changes in TGF-beta2 mRNA levels, twice as much TGF-beta2 protein was measured in the kidney by day 30 of STZ-induced diabetes compared with day 0 controls analyzed by Western blotting (P < 0.05), and the protein was localized both to the nuclei and cytoplasm of glomerular cells, analyzed by immunocytochemistry. In addition, three times as much TGF-betaRII protein was found by day 90 of STZ-induced diabetes compared with day 0 controls, making this the most responsive receptor type. These results suggest that the entire TGF-beta axis has a role in the etiology of kidney fibrosis and could be manipulated therapeutically to preserve kidney function.

The Renal Expression of Transforming Growth Factor- Isoforms and Their Receptors in Acute and Chronic Experimental Diabetes in Rats

Transforming growth factors-β (TGF-β) are fibrogenic factors that have been strongly implicated in the development of diabetic nephropathy. Our aim was to use two animal models [the streptozotocin (STZ)-induced diabetic rat and the genetically prone biobreeding (BB) rat] to fully characterize the responses of the renal TGF-β system in both short- and long-term diabetes. In this study changes in the entire renal TGF-β system, at both protein and messenger RNA (mRNA) levels, have been characterized using the techniques of immunocytochemistry, Western blotting, and ribonuclease protection assay. We also used Western blotting of pro-collagen-I C-peptide to demonstrate that the rate of fibrogenesis was highest over the first 2 weeks of diabetes. TGF-β1, TGF-β2, and receptor mRNA and protein were detected in the control nondiabetic kidney. It was found that dramatic and dynamic changes occur in all parts of the renal TGF-β axis in both models of experimental diabetes, but TGF-β2 and TGF-βRII proteins were the predominant responsive element, particularly during the acute phase of disease. For example, during the acute phase of disease (0–30 days), although renal TGF-β1 mRNA levels were elevated, no increases in the corresponding protein were detected in the kidney. By contrast, in the absence of changes in TGF-β2 mRNA levels, twice as much TGF-β2 protein was measured in the kidney by day 30 of STZ-induced diabetes compared with day 0 controls analyzed by Western blotting (P < 0.05), and the protein was localized both to the nuclei and cytoplasm of glomerular cells, analyzed by immunocytochemistry. In addition, three times as much TGF-βRII protein was found by day 90 of STZ-induced diabetes compared with day 0 controls, making this the most responsive receptor type. These results suggest that the entire TGF-β axis has a role in the etiology of kidney fibrosis and could be manipulated therapeutically to preserve kidney function.

Prediction of Diabetes in Biobreeding/Aburahi Rats by the Measurement of Soluble L-Selectin

L-selectin was initially identified as a homing receptor, recently soluble L-selectin has been used as a marker of the inflammation. Therefore, we investigated the relation between the development of diabetes and serum L-selectin levels in the Biobreeding (BB) rats. Serum L-selectin were measured from 30 days old to the onset of diabetes or to 90 days old in Biobreeding (BB) rats and Wistar Furth (WF) rats. Significant elevation of L-selectin was found in diabetes prone (DP) rats from 45 days old to the onset of diabetes or through 90 days old. No elevation was found in other strain of rats. In histological study, all of DP rats had insulitis and no other strain of rats had it. Therefore, we conclude that the measurement of serum L-selectin could be useful tool to predict the onset of diabetes or presence of insulitis in BB rats

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.

Elevated apoptosis of peripheral T lymphocytes in diabetic BB rats.

Thymocytes and peripheral lymphocytes of BioBreeding (BB) diabetes-prone (BBDP) and diabetes-resistant (BBDR) rat were analysed by fluorescence-activated cell sorter (FACS). The number of CD4- CD8-, CD4+ CD8-, CD4- CD8+ and CD4+ CD8+ subsets was not different between BBDP and BBDR rat thymocytes, whereas spleen and lymph nodes in BBDP rats undergo severe T-cell lymphopenia. Notably, mature CD4- CD8+ [T-cell receptor (TCR)-alphabeta+ and CD5+] cells are certainly present in the BBDP rat thymus, unlike some previous reports, suggesting that the differentiation of CD4- CD8+ from CD4+ CD8+ cells occurs normally in the BBDP rat thymus. As a cause of peripheral T-cell lymphopenia we suspected apoptosis of recent thymic emigrants. By FACS analysis with fluorescein isothiocyanate-labelled annexin V, elevated apoptosis was evident in BBDP rat peripheral lymphocytes. Furthermore, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling (TUNEL) staining in BBDP rat splenic sections revealed that a number of TUNEL-positive cells were observed in the T-lymphocyte-rich area. From these results, we postulate that an abnormally elevated apoptosis of peripheral T lymphocytes, but not impaired thymocyte differentiation, is a cause of the peripheral T-cell lymphopenia in BBDP rats.

Cellular and molecular roles of beta cell autoantigens, macrophages and T cells in the pathogenesis of autoimmune diabetes.

Type I diabetes, also known as insulin-dependent diabetes mellitus (IDDM) results from the destruction of insulin-producing pancreatic beta cells by a progressive beta cell-specific autoimmune process. The pathogenesis of autoimmune IDDM has been extensively studied for the past two decades using animal models such as the non-obese diabetic (NOD) mouse and the BioBreeding (BB) rat. However, the initial events that trigger the immune responses leading to the selective destruction of the beta cells are poorly understood. It is thought that beta cell autoantigens are involved in the triggering of beta cell-specific autoimmunity. Among a dozen putative beta cell autoantigens, glutamic acid decarboxylase (GAD) has been proposed as perhaps the strongest candidate in both humans and the NOD mouse. In the NOD mouse, GAD, as compared with other beta cell autoantigens, provokes the earliest T cell proliferative response. The suppression of GAD expression in the beta cells results in the prevention of autoimmune diabetes in NOD mice. In addition, the major populations of cells infiltrating the islets during the early stage of insulitis in BB rats and NOD mice are macrophages and dendritic cells. The inactivation of macrophages in NOD mice results in the prevention of T cell mediated autoimmune diabetes. Macrophages are primary contributors to the creation of the immune environment conducive to the development and activation of beta cell-specific Th1-type CD4+ T cells and CD8+ cytotoxic T cells that cause autoimmune diabetes in NOD mice. CD4+ and CD8+ T cells are both believed to be important for the destruction of beta cells. These cells, as final effectors, can kill the insulin-producing beta cells by the induction of apoptosis. In addition, CD8+ cytotoxic T cells release granzyme and cytolysin (perforin), which are also toxic to beta cells. In this way, macrophages, CD4+ T cells and CD8+ T cells act synergistically to kill the beta cells in conjunction with beta cell autoantigens and MHC class I and class II antigens, resulting in the onset of autoimmune type I diabetes.

BB rat diabetes susceptibility and body weight regulation genes colocalize on chromosome 2.

The genetic etiology of Type 1 (insulin-dependent) diabetes mellitus is complicated by the apparent presence of several diabetes susceptibility genetic regions. Type 1 diabetes in the inbred BioBreeding (BB) rat closely resembles the human disorder and was previously shown to involve two genes: the lymphopenia (lyp) region on Chromosome (Chr) 4 and RT1(u) in the major histocompatibility complex (MHC) on Chr 20. In addition, a segregation analysis of an F(2) intercross between the diabetes-prone congenic BB DR(lyp/lyp, u/u) and F344(+/+,)(lv/lv) rats indicated that at least one more genetic factor was responsible for Type 1 diabetes. In this study, we generated F(2)N(2) progeny in a cross between non-diabetic F(2)(DR(lyp/lyp,u/u) x F344)(lyp/lyp,u/u) and diabetic DR(lyp/lyp, u/u) rats. In a subsequent total genome scan, a third factor was mapped to the 21.3-cM region on Chr 2 between D2Mit14 and D2Mit15 (peak LOD score 4.7 with 67% penetrance). Interestingly, the homozygosity of the BB allele (b/b) for the Chr 2 region was significantly associated with a greater weight reduction after fasting than the homozygosity of the F344 allele (f/f, p < 0.008). In conclusion, the development of Type 1 diabetes in the congenic DR(lyp/lyp) rat is controlled by at least three genes: lymphopenia, MHC, and a third factor that may play a role in metabolism and body weight regulation.

The NOD mouse model of type 1 diabetes: as good as it gets?

Of the two well-known rodent models of human type 1 diabetes (the non-obese diabetic mouse and the BioBreeding rat) the mouse has become the model of choice. Here the authors re-examine the value of this mouse model as a tool for understanding human diabetes and for testing potential therapies, reviewing both strengths and weaknesses.

A Diabetogenic Gene Prevents T Cells from Receiving Costimulatory Signals

T cell fate following antigen encounter is determined by several intracellular signals generated by the interaction of the T cell with an antigen-presenting cell. In the periphery activation requires T cell receptor signaling (signal one) in combination with costimulatory signals (signal two), usually provided through the cognate interaction of CD28 and B7 molecules. Provision of signal one alone to purified murine peripheral T cells in vitro induces apoptosis or anergy rather than promoting activation. These T cells can be rescued from apoptosis if they are provided with costimulation supplied, for example, by engaging the CD28 co-receptor with an anti-CD28 monoclonal antibody or by adding an exogenous source of interleukin-2. However, a majority of peripheral T cells from autoimmune, diabetes-prone Biobreeding (BB) rats exhibited different responses to these stimuli. T cells from these rats could not be rescued from apoptosis by costimulation. This was not due to the inability of BB-DP T cells to upregulate CD28 and the IL-2 receptor in response to TCR crosslinking. The failure of these costimulatory interactions to rescue BB-DP T cells segregated with the diabetes-susceptibility gene iddm1. Iddm1 in the rat causes peripheral T cell lymphopenia, which is associated with a dramatically shortened peripheral T cell life span. Our results indicate that a diabetogenic gene may contribute to autoimmunity by negating costimulatory signals important for the survival of long-lived peripheral T cells.

Recapitulation of Normal and Abnormal BioBreeding Rat T Cell Development in Adult Thymus Organ Culture

Congenitally lymphopenic diabetes-prone (DP) BioBreeding (BB) rats develop spontaneous T cell-dependent autoimmunity. Coisogenic diabetes-resistant (DR) BB rats are not lymphopenic and are free of spontaneous autoimmune disease, but become diabetic in response to depletion of RT6+ T cells. The basis for the predisposition to autoimmunity in BB rats is unknown. Abnormal T cell development in DP-BB rats can be detected intrathymically, and thymocytes from DR-BB rats adoptively transfer diabetes. The mechanisms underlying these T cell developmental abnormalities are not known. To study these processes, we established adult thymus organ cultures (ATOC). We report that cultured DR- and DP-BB rat thymi generate mature CD4 and CD8 single-positive cells with up-regulated TCRs. DR-BB rat cultures also generate T cells that express RT6. In contrast, DP-BB rat cultures generate fewer CD4+, CD8+, and RT6+ T cells. Analysis of the cells obtained from ATOC suggested that the failure of cultured DP-BB rat thymi to generate T cells with a mature phenotype is due in part to an increased rate of apoptosis. Consistent with this inference, we observed that addition of the general caspase inhibitor Z-VAD-FMK substantially increases the number of both mature and immature T cells produced by DP-BB rat ATOC. We conclude that cultured DR-BB and DP-BB rat thymi, respectively, recapitulate the normal and abnormal T cell developmental kinetics and phenotypes observed in these animals in vivo. Such cultures should facilitate identification of the underlying pathological processes that lead to immune dysfunction and autoimmunity in BB rats.

Both CD4+and CD8+T cells are Required for IFN-γ Gene Expression in Pancreatic Islets and Autoimmune Diabetes Development in Biobreeding Rats

To study the relative roles of CD4+and CD8+T cells and their cytokine products in autoimmune diabetes development, we selectively depleted CD4+and CD8+T cells in autoimmune diabetes-prone (DP) biobreeding (BB) rats, by administrations of anti-CD2 and anti-CD8 monoclonal antibody (mAb) respectively. We then analysed cytokine mRNA expression, by PCR assay, in mononuclear leukocytes isolated from islets and spleens of control and mAb-treated DP-BB rats. Depletion of CD4+T cells (by anti-CD2mAb) in blood, spleen and islets prevented diabetes development in DP-BB rats, and depletion of CD8+T cells (by anti-CD8mAb) delayed and significantly decreased diabetes incidence. Depletion of either CD4+or CD8+T cells completely prevented IFN-γ mRNA upregulation in islets of DP-BB rats above the low level expressed in islets of diabetes-resistant (DR) BB rats. Also, IL-10mRNA levels in islets of DP-BB rats were significantly decreased by depletion of either CD4+or CD8+T cells, whereas the effects of the anti-T cell mAb on mRNA levels of other cytokines in islets (IL-2, IL-4, IL-12p40, and TNF-α) were discordant. In contrast, both mAb treatments significantly upregulated IL-4 and TNF-α mRNA levels in spleens of DP-BB rats. These results demonstrate that islet infiltration by both CD4+and CD8+T cells is required for IFN-γ and IL-10 production in islets and β-cell destruction. Depletion of either CD4+or CD8+T cells may prevent β-cell destruction by decreasing IFN-γ and IL-10 production in islets and increasing IL-4 and TNF-α production systemically.

Differential Expression of p95vav in Primary Lymphoid Tissue of BB Rats Congenic for the Lymphopenia Gene

Lyp controls lymphopenia and T-cell mediated autoimmune diabetes in the Bio Breeding (BB) rat possibly by interacting with T-cell maturation in the thymus. The protooncogene vav (p95) is involved in T-cell activation and in the intrathymic selection of developing T cells. We have previously reported increased production of IFN-γ of self reactive thymocytes in the thymus medulla of Lyp/Lyp BB rats. Lymphopenia and diabetes may therefore be linked to an increase in thymocyte activation leading to a bias in thymocyte development. The purpose of this study was therefore to investigate whether the expression of p95vav in primary lymphoid tissues from congenic Lyp/Lyp, Lyp/+ and +/+ BB rats was correlated to the Lyp genotype using in situ hybridization and reverse transcription (RT)-PCR. It was found that the expression of vav mRNA in the thymus was increased in Lyp/Lyp compared to Lyp/+ and +/+ rats (p < 0.05). Western blot analysis revealed that the amount of p95vav protein in Lyp/Lyp thymus was also increased. The results show that vav expression correlates with the lymphopenia phenotype and diabetes development in congenic Lyp/Lyp BB rats. An increase in the availability of p95vav during the development and activation of thymocytes in Lyp/Lyp BB rats may therefore contribute to the generation of islet autoreactivity, lymphopenia or both.

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.

Immunisation and type 1 diabetes mellitus: is there a link?

Recent evidence from animal studies has raised the possibility that immunisation by vaccines can influence the pathogenesis of type I (insulin-dependent) diabetes mellitus. In non-obese diabetic mice and biobreeding rats, complete Freund's adjuvant and bacillus Calmette-Guérin (BCG) vaccine have successfully been used to interrupt the development of diabetes mellitus. This effect is probably mediated by nonspecific suppression of the autoimmune process. A number of attempts have also been made to assess the impact of parenteral immunisation on type 1 diabetes mellitus in humans. Epidemiological evidence has not indicated any clear link between BCG vaccination and the development of diabetes mellitus in humans. Some reports have suggested that natural mumps or mumps vaccinations can induce islet cell autoimmunity, but there is no evidence that mumps-measles-rubella mass vaccination programmes have changed the incidence of diabetes mellitus in any population. An independent protective role of measles virus has been suggested in one study. Recent studies have indicated that enterovirus infections may induce beta cell autoimmunity and clinical diabetes. The only currently available enterovirus vaccine is the poliovirus vaccine which, in theory, could modulate the protection against other enteroviruses by inducing cross-reactive T cell immune responses; however, this hypothesis has not been tested so far. In conclusion, there is no clear evidence that any currently used vaccine can prevent or induce diabetes in humans. However, only a few studies are available on the subject and therefore the possibility of a link between vaccination and diabetes mellitus cannot be excluded.

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

Signs of Immaturity of Splenic Dendritic Cells from the Autoimmune Prone Biobreeding Rat: Consequences for the In Vitro Expansion of Regulator and Effector T Cells

From the biobreeding-diabetic prone (BB-DP) rat, an animal model for endocrine autoimmunity, phenotype and function of splenic dendritic cells (DC) were studied. Furthermore, the suppressive effect of peritoneal macrophages (pMphi) from the BB-DP rat in the MLR was investigated. Lower numbers of splenic DC were isolated from BB-DP rats than from control Wistar rats. In the preautoimmune phase, DC of the BB-DP rat had a lower surface MHC class II expression (and in preliminary data, a lower CD80 expression), ingested more bacteria, and had a lower stimulatory potency in the syngeneic (syn)MLR as compared with control DC. During disease development, the MHC class II expression further decreased, and a low stimulatory activity became evident in the allogeneic (allo)MLR. With regard to the expansion of suppressor/regulatory T cells, a lower percentage of RT6+ T cells but higher percentages of CD45RClow T cells were induced by BB-DP DC in synMLR, but not in alloMLR. An increase in the CD4/CD8 T cell ratio was observed in both the syn- and alloMLR due to a relative weak expansion of CD8+ T cells with DC of the BB-DP rat. Resident pMphi isolated from BB-DP or Wistar rats were equally effective in suppressing the DC-driven synMLR. In conclusion, splenic DC from the BB-DP rat have a lower accessory cell function already at young age, before the development of disease, and expanded different subsets of effector/suppressor T cells in vitro as compared with those from Wistar rats. The dysfunction of DC from BB-DP rats is likely to be caused by their relative immaturity as indicated by their low class II and costimulatory molecule expression and relatively high phagocytic activity.

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.