Induction of glutamic acid decarboxylase 65-specific Th2 cells and suppression of autoimmune diabetes at late stages of disease is epitope dependent.

Peptide-based immunotherapy is one strategy by which to selectively suppress the T cell-mediated destruction of beta cells and treat insulin-dependent diabetes mellitus (IDDM). Here, we investigated whether a panel of T cell epitopes derived from the beta cell autoantigen glutamic acid decarboxylase 65 (GAD65) differ in their capacity to induce Th2 cell function in nonobese diabetic (NOD) mice and in turn prevent overt IDDM at different preclinical stages of disease development. The panel consists of GAD65-specific peptides spanning aa 217-236 (p217), 247-265 (p247), 290-309 (p290), and 524-543 (p524). Our studies revealed that all of the peptides effectively prevented insulitis and diabetes when administered to NOD mice before the onset of insulitis. In contrast, only a mixture of p217 and p290 prevented progression of insulitis and overt IDDM in NOD mice exhibiting extensive beta cell autoimmunity. Immunization with the GAD65-specific peptides did not block IDDM development in NOD mice deficient in IL-4 expression. These findings demonstrate that GAD65-specific peptide immunotherapy effectively suppresses progression to overt IDDM, requires the production of IL-4, and is dependent on the epitope targeted and the extent of preexisting beta cell autoimmunity in the recipient.     

Autoimmunity-prone BB rats lack functional cytotoxic T cells

BB rats are prone to develop an autoimmune form of insulin-dependent diabetes mellitus (IDDM) and thyroiditis. Development of autoimmunity is thymus dependent. Previous studies have shown that BB rats lack a population of T cells bearing the RT6 antigen and have very low numbers of suppressor/cytotoxic T cells. In this study, we confirm that BB rats have decreased numbers of phenotypic T suppressor/cytotoxic (Ts/c) cells (OX19+, OX8+ cells) in their lymphoid organs. Moreover, we find that the phenotypic Ts/c cells of BB rats lack apparent cytotoxic activity. These T cells fail to kill allogeneic target cells in a cell-mediated lympholysis assay and fail to generate lectin-dependent cytotoxicity. The addition of interleukin 2, gamma-interferon, and other lymphokines to cultures of BB T cells does not induce functional cytotoxic T lymphocytes. We find that the activated T cells of newly diabetic rats are incapable of killing major-histocompatibility-complex-matched islet cells, despite the ability of these cells to cause IDDM in passive transfer experiments. We conclude that autoimmune disease occurs in BB rats in the absence of functional cytotoxic T cells.     

Delayed remodeling in the early period of fracture healing in spontaneously diabetic BB/OK rats depending on the diabetic metabolic state

Several clinical series, analyzing fracture healing in patients with insulin-dependent type 1 diabetes (IDDM) demonstrated significant incidence of delayed union, non-union, and pseudarthrosis. The purpose of this study was to examine the detailed histomorphometry and histology of bone formation and remodeling during fracture healing depending on the diabetic metabolic state in spontaneously diabetic BB/O(ttawa)K(arlsburg) rats, a rat strain that represents a close homology to IDDM in man. A standardized fracture model was chosen and based on blood-glucose values at the time of surgery (mg%), postoperative blood-glucose course (mg%) and postoperative insulin requirements (IU/kg), 100 spontaneously diabetic BB/OK rats were divided into groups with well-compensated (n=50, 167+/-77 mg%; 244+/-68 mg%; 1.8+/-1.9 IU/kg) or poorly compensated (n=50, 380+/-89 mg%; 415+/-80 mg%; 6.0+/-1.0 IU/kg) metabolic state. Fifty LEW.1A rats served as the normoglycemic controls (97+/-15 mg%). Ten animals from each group were killed 1, 2, 3, 4 and 6 weeks after fracture and specimens were processed undecalcified for quantitative histomorphometry and for qualitative light microscopy. In terms of bone histomorphometry, within the first four weeks after fracture, severe mineralization disorders occurred exclusively in the rats with poorly compensated diabetic metabolic states with a significantly decrease of all fluorochrome-based parameters of mineralization, apposition, formation and timing of mineralization in comparison to the spontaneously diabetic rats with well-compensated metabolic states and to the control rats. This was confirmed histologically. Early fracture healing in the spontaneously diabetic BB/OK rats is delayed exclusively in poorly compensated diabetic metabolic states, and 6 weeks after fracture, histomorphometrically significant deficits in the measured and dynamically calculated parameters remain. This study suggests that strictly controlled insulin treatment resulting in well-compensated diabetic metabolic states will ameliorate the impaired early mineralization and cell differentiation disorders of IDDM fracture healing.     

Molecular characterization of MHC class II antigens (β 1 domain) in the BB diabetes-prone and -resistant rat

The BB or BB/Worcester (BB/W) rat is widely recognized as a model for human insulin-dependent diabetes mellitus (IDDM). Of at least three genes implicated in genetic susceptibility to IDDM in this strain, one is clearly linked to the major histocompatibility complex (MHC). In an attempt to define the diabetogenic gene(s) linked to the MHC of the BB rat, cDNA clones encoding the class II MHC gene products of the BB diabetes-prone and diabetes-resistant sublines have been isolated and sequenced. For comparison, the ₁ domain of class II genes of the Lewis rat (RTl^L) were sequenced. Analysis of the sequence data reveals that the first domain of RT1.D and RT1.B chain of the BB rat are different from other rat or mouse class 11 sequences. However, these sequences were identical in both the BB diabetes-prone and BB diabetes-resistant sublines. The significance of these findings is discussed in relation to MHC class II sequence data in IDDM patients and in the nonobese diabetic (NOD) mouse strain.     

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.     

Immune attack on pancreatic islet transplants in the spontaneously diabetic BioBreeding/Worcester (BB/W) rat is not MHC restricted

Spontaneous diabetes mellitus in the BB/W rat is preceded by lymphocytic insulitis that destroys pancreatic beta cells. Cultured pancreatic islets and adrenal cortex from inbred rats of variable MHC were transplanted to RT1/u BB/W rats without allograft rejection. Islet grafts from RT1/u and non-RT1/u rats evidenced lymphocytic insulitis in BB/W recipients that became diabetic or evidenced lymphocytic insulitis within endogenous islets. These findings suggest that BB immune insulitis is not MHC restricted and may be directed against islet transplants from non-RT1/u animals.     

Functional status of the immune system after chronic administration of 2'-deoxycoformycin in the BB rat

Insulin-dependent diabetes mellitus (IDDM) is caused by autoimmune destruction of pancreatic beta cells with the primary mechanism being cell mediated. The BB rat develops insulitis and IDDM with many features analogous to the disease in man. In previous studies we reported that weekly administration of 2'-deoxycoformycin (dCF) for four months reduces significantly the incidence of IDDM in the BB rat by 70%, and that the animals remain free of diabetes for a minimum of two months after drug withdrawal. Since the diabetes-prone BB rat is lymphopenic, with a reduction of both CD4 and CD8 cells, the continuous failure of dCF treated animals to develop diabetes may have been due to generalized immunosuppression. To test this possibility, the ability of dCF treated diabetes-free BB rats to mount an immune response after challenge with Ovalbumin was examined five months after drug withdrawal. The results showed that the post-immunization levels of total IgG and specific IgG in these animals did not differ from those observed in non-dCF treated controls nor those of control diabetes-resistant non-lymphopenic BB rats. Moreover, FACS analysis indicated no change in the percentages of total numbers of CD4+ or CD8+ cells between the two groups of animals. Histological assessment of the pancreata of the post-dCF treated animals showed varying degrees of mononuclear cell infiltrates in the islets. These data demonstrate that treatment by dCF is not permanent, and may require intermittent or continuous administration to prevent development of diabetes. Further studies are needed to determine the mechanism of action of dCF in this model of IDDM.     

Congenic diabetes-prone BB.Sa and BB.Xs rats differ from their progenitor strain BB/OK in frequency and severity of insulin-dependent diabetes mellitus.

Two newly established congenic diabetes-prone BB rat strains designated BB.Sa and BB.Xs carrying a region of chromosome 1 (Sa-Lsn-Secr-Igf2-Tnt, 16 cM) and a region of chromosome X (DXMgh3-Mycs/Pfkb1-Ar, 36 cM) of the SHR rats, respectively, were studied to determine whether the transferred chromosomal regions influence diabetes frequency, age at onset, and clinical picture. Therefore, 4 complete litters of BB/OK (n = 43), BB.Sa (n = 45), and BB.Xs (n = 41) were observed for diabetes occurrence up to the age of 30 weeks. From these litters 6 diabetic males of each strain manifesting in an interval of 1 week were chosen to study body weight, blood glucose, insulin requirement to survive, and several diabetes-related serum constituents at onset of diabetes and after a diabetes duration of 150 days. The diabetes frequency was significantly lower in BB.Xs than in rats of the parental strain BB/OK, whereas comparable frequencies were found between BB/OK and BB.Sa rats. Obvious differences were observed 150 days after diabetes onset between BB/OK and both BB.Sa and BB.Xs rats. BB/OK rats were significantly heavier and needed significantly more insulin/100 g body weight than BB.Sa and BB.Xs rats. Comparisons of the serum constituents as lipids, proteins, and minerals revealed significant differences between diabetic BB/OK rats and their diabetic congenic derivatives in several traits studied at onset and after 150 days of insulin treatment. These results not only show the power of congenic lines in diabetes research, but indicate for the first time that there are genetic factors on chromosomes 1 and X influencing frequency and severity of diabetes in the BB/OK rat.     

Abrogation of diabetes in BB rats by acute virus infection. Association of viral-lymphocyte interactions

The BB rat spontaneously develops an insulin-dependent diabetes mellitus (IDDM) that closely resembles this disease in man. The pathogenesis involves autoimmune destruction of pancreatic beta-cells. In the present study, inoculation of diabetes-prone BB rats at 30 days of age with a lymphotropic variant of lymphocytic choriomeningitis virus significantly reduced the incidence of diabetes. Such virus-inoculated, diabetes-free rats had normal levels of pancreatic insulin and little or no mononuclear cell infiltration in the islets. Virus was recovered from lymphocytes by cocultivation with permissive cells. In contrast, virus was not detected in a wide variety of organs, indicating that infection in BB rats was primarily lymphotropic. PBL analyzed by FACS and monoclonal markers showed a marked reduction of pan-T. Th, and T suppressor/cytotoxic lymphocyte subsets restricted to 4 and 7 days after infection when compared with numbers of lymphocytes in uninoculated diabetes-prone rats. To prevent IDDM, replicating virus was required, because the expected incidence of IDDM in diabetes prone rats inoculated with UV-inactivated virus was equivalent to that of untreated animals. These results suggest that a virus can suppress the autoimmune response that would otherwise have caused IDDM and may be useful as a probe in dissecting the molecular basis of this autoimmune disorder.     

A study of hepatic metabolism of thyroxine in BB/W rats treated with L-thyroxine

The effect of thyroxine (T4) on T4 conversion to triiodothyronine (T3) and reverse T3 (rT3) was studied in BB/W rats. A colony of 38 BB/W rats was obtained and half were treated with thyroxine (T4), 1 mg per liter of drinking water. At 106 days of age the following groups were identified: nondiabetic, no T4 treatment, 8 rats; nondiabetic, T4 treated, 8 rats; diabetic, no T4 treatment, 10 rats; diabetic, T4 treated, 7 rats. All animals with diabetes were treated with insulin. T4 conversion to T3 and rT3 was assessed in liver homogenates in 0.1 M Tris-HCl buffer, pH 7.4, with or without 5 mM dithiothreitol (DDT). Serum T4 and rT3 were significantly elevated in both T4-treated groups (P less than 0.001), while serum T3 was not affected in either. Basal T4 deiodination to T3 by the liver homogenate did not change on treatment with T4; the addition of DTT increased T3 production in the homogenate from T4 treated nondiabetic animals (P less than 0.05). In both nondiabetic and insulin-treated diabetic rats there was no effect of T4 on the rate of rT3 production. Since, in the rat, 30-40% of circulating T3 is a direct contribution of thyroid gland secretion, and that would be absent in our T4-suppressed animals, the normal serum T3 may reflect increased absolute peripheral T3 production from the greater concentration of circulating T4.     

Genes of SHR rats protect spontaneously diabetic BB/OK rats from diabetes: lessons from congenic BB.SHR rat strains

Diabetes in BB rats share many common features with human type 1 diabetes. One of them is the complex and polygenic nature of disease. Analysis of cross hybrids of diabetic BB/OK rats and rats of different diabetes-resistant strains has demonstrated that beside the MHC genes, Iddm1 and the lymphopenia, Iddm2, additional non-MHC genes are involved in diabetes development. To study the importance of the non-MHC genes, Iddm4 and Iddm3, two congenic BB.SHR rat strains were generated by recombining a segment of the SHR chromosome 6 (Iddm4; termed BB.6S; 15cM) or chromosome 18 (Iddm3; termed BB.18S; 24cM) into the BB/OK background by serial backcrossing and marker-aided selection. The characterization of both congenic strains demonstrates a drastic reduction of diabetes frequency in comparison to the BB/OK strain (86% vs 14% and 34%). It is supposed that diabetes protective genes of SHR must be located on both chromosomal segments and that these suppress the action of the essential and most important genes of diabetes development in the BB/OK rat, Iddm1, and Iddm2.     

Adenoviral insulin gene therapy prolongs survival of IDDM model BB rats by improving hyperlipidemia.

Diabetes is frequently associated with hyperlipidemia, which results in atherogenic complications. Insulin-dependent diabetes mellitus (IDDM) model BB/Wor//Tky (BB) rats exhibit both hyperglycemia and hyperlipidemia and die within 3 weeks after the onset of diabetes unless insulin therapy is given. We performed insulin gene therapy in BB rats with adenovirus vectors through the tail vein. After infusion, plasma triglyceride levels dropped quickly and maintained low levels for 1 week, whereas blood glucose levels showed a slight decrease. The survival period of diabetic BB rats was prolonged to up to 75 days by infusing insulin gene-expressing adenoviral vectors. We suggest that the control of hyperlipidemia can be a life-saving measure when combined with hyperglycemia control in the treatment of diabetes.     

Endothelins urinary excretion is increased in spontaneously diabetic rats: BB/BB

Previously we reported (1) an increase of endothelin-1,2 (ET) content, in urine of rats made diabetic with streptozotocin (STZ), starting three days and up to 20 weeks from diabetes induction. The increased ET excretion was considered as an early marker of endothelial damage. To ascertain if this phenomenon was present also in a strain of spontaneously diabetic rats, endothelin-1,2 urinary excretion was determined in BB/BB diabetic rats, and their control (BB/WB), at different times after the onset of diabetes, (two, four, six and twelve weeks). BB/BB diabetic rats showed elevated urinary excretion of endothelins as compared to BB/WB control rats, starting two weeks after diabetes onset, and up to twelve weeks. In the same animals, Nerve Conduction Velocity (NCV), was monitored at the same time as an index of the occurrence of a diabetes complication (peripheral neuropathy). NCV resulted to be impaired in the BB/BB diabetic rats as compared to control rats; however the increase of ET in urine, is earlier in comparison to peripheral neuropathy. These data suggest the hypothesis that endothelial damages preceed the overt manifestations of peripheral neuropathy associated to diabetes.     

Seven regions of the genome show evidence of linkage to type 1 diabetes in a consensus analysis of 767 multiplex families

Type 1 diabetes (T1D) is a genetically complex disorder of glucose homeostasis that results from the autoimmune destruction of the insulin-secreting cells of the pancreas. Two previous whole-genome scans for linkage to T1D in 187 and 356 families containing affected sib pairs (ASPs) yielded apparently conflicting results, despite partial overlap in the families analyzed. However, each of these studies individually lacked power to detect loci with locus-specific disease prevalence/sib-risk ratios (lambda(s)) <1.4. In the present study, a third genome scan was performed using a new collection of 225 multiplex families with T1D, and the data from all three of these genome scans were merged and analyzed jointly. The combined sample of 831 ASPs, all with both parents genotyped, provided 90% power to detect linkage for loci with lambda(s) = 1.3 at P=7.4x10(-4). Three chromosome regions were identified that showed significant evidence of linkage (P<2.2x10(-5); LOD scores >4), 6p21 (IDDM1), 11p15 (IDDM2), 16q22-q24, and four more that showed suggestive evidence (P<7.4x10(-4), LOD scores > or =2.2), 10p11 (IDDM10), 2q31 (IDDM7, IDDM12, and IDDM13), 6q21 (IDDM15), and 1q42. Exploratory analyses, taking into account the presence of specific high-risk HLA genotypes or affected sibs' ages at disease onset, provided evidence of linkage at several additional sites, including the putative IDDM8 locus on chromosome 6q27. Our results indicate that much of the difficulty in mapping T1D susceptibility genes results from inadequate sample sizes, and the results point to the value of future international collaborations to assemble and analyze much larger data sets for linkage in complex diseases.     

Abnormal thymocyte maturation in spontaneously diabetic BB rats involves the deletion of CD4-8+ cells.

The object of this study was to further characterize the pathophysiology of the peripheral T lymphopenia in the BB rat. Towards this end, surface markers on unseparated thymocytes and purified thymocyte subsets from age- and sex-matched diabetes-resistant (BBn) and diabetes-prone (BBd) rats were analyzed by two-color flow cytometry. The proportions of thymocytes falling into each of the four main phenotypic subsets were comparable in BBn (n = 9) and BBd (n = 8) rats: respectively, 4.6 +/- 0.6% and 4.4 +/- 0.8%, CD4-8-; 68.1 +/- 1.9% and 71.1 +/- 3.2%, CD4+8+; 18.3 +/- 1.5% and 15.4 +/- 2.3%, CD4+8-; 9.1 +/- 0.9% and 9.1 +/- 1.0%, CD4-8+. In addition, absolute numbers of thymocytes were not significantly different. The levels of expression of CD4, TCR-alpha beta within each thymocyte subset were comparable in BBn and BBd animals as were the anti-TCR-induced proliferative responses of their CD4+8- and CD4-8+ thymocytes. However, phenotypic abnormalities within the CD4-8+ thymocyte subset of the BBd rat were found. A very significant (p less than 0.005) deletion of mature CD4-8+, TCR-alpha beta + thymocytes and a proportional increase (p less than 0.005) of immature CD4-8+, TCR-alpha beta low thymocytes. Moreover, a twofold decrease of CD8 expression by mature CD4-8+ thymocytes was observed in BBd animals. These results suggest that an impaired thymic maturation contributes to the peripheral T lymphopenia of the BBd rat.     

Diabetic hypertriglyceridaemia and related 5' flanking polymorphism of the human insulin gene.

A polymorphic DNA sequence was studied on the 5'' flanking region of the human insulin gene in relation to diabetic lipaemia. The genotype frequencies in a control population (n = 52) were homozygous L 6%, heterozygous 54%, and homozygous S 40%. Corresponding genotype frequencies in a hypertriglyceridaemic group (n = 74) were 18%, 66%, and 16% (p less than 0.01; chi 2 test). When the hypertriglyceridaemic patients were divided on the basis of glucose tolerance the corresponding genotype frequencies in the diabetic subgroup (n = 23) were 39%, 52%, and 9% compared with 0%, 74%, and 26% in the non-diabetics (n = 34) (p less than 0.001; chi 2 test). These findings suggest that the homozygous L genotype may confer susceptibility to diabetic hypertriglyceridaemia.     

Comparison of non-mydriatic retinal photography with ophthalmoscopy in 2159 patients: mobile retinal camera study.

OBJECTIVE--To determine whether non-mydriatic Polaroid retinal photography was comparable to ophthalmoscopy with mydriasis in routine clinic screening for early, treatable diabetic retinopathy. DESIGN--Prospective study of ophthalmoscopic findings according to retinal camera screening and ophthalmoscopy and outcome of referral to ophthalmologist. SETTING--Outpatient diabetic clinics of three teaching hospitals and three district general hospitals. PATIENTS--2159 Adults selected randomly from the diabetic clinics, excluding only those registered as blind or those in wheelchairs and unable to enter the screening vehicle. MAIN OUTCOME MEASURES--Numbers of patients and eyes correctly identified by each technique as requiring referral with potentially treatable retinopathy (new vessel formation and maculopathy) and congruence in numbers of microaneurysms, haemorrhages, and exudates reported. RESULTS--Camera screening missed two cases of new vessel formation and did not identify a further 12 but indicated a need for referral. Ophthalmoscopy missed five cases of new vessel formation and indicated a need for referral in another four for other reasons. Maculopathy was reported in 147 eyes with camera screening alone and 95 eyes by ophthalmoscopy only (chi 2 = 11.2; p less than 0.001), in 66 and 29 of which respectively maculopathy was subsequently confirmed. Overall, 38 eyes received laser treatment for maculopathy after detection by camera screening compared with 17 after ophthalmoscopic detection (chi 2 = 8.0; p less than 0.01). Camera screening underestimated numbers of microaneurysms (chi 2 = 12.9; p less than 0.001) and haemorrhages (chi 2 = 7.4; p less than 0.01) and ophthalmoscopy underestimated hard exudates (chi 2 = 48.2; p less than 0.001). CONCLUSIONS--Non-mydriatic Polaroid retinal photography is at least as good as ophthalmoscopy with mydriasis in routine diabetic clinics in identifying new vessel formation and absence of retinopathy and is significantly better in detecting exudative maculopathy.