Results, meta-analysis and a first evaluation of UNOxR, the urinary nitrate-to-nitrite molar ratio, as a measure of nitrite reabsorption in experimental and clinical settings.

We recently found that renal carbonic anhydrase (CA) is involved in the reabsorption of inorganic nitrite (NO2-), an abundant reservoir of nitric oxide (NO) in tissues and cells. Impaired NO synthesis in the endothelium and decreased NO bioavailability in the circulation are considered major contributors to the development and progression of renal and cardiovascular diseases in different conditions including diabetes. Isolated human and bovine erythrocytic CAII and CAIV can convert nitrite to nitrous acid (HONO) and its anhydride N2O3 which, in the presence of thiols (RSH), are further converted to S-nitrosothiols (RSNO) and NO. Thus, CA may be responsible both for the homeostasis of nitrite and for its bioactivation to RSNO/NO. We hypothesized that enhanced excretion of nitrite in the urine may contribute to NO-related dysfunctions in the renal and cardiovascular systems, and proposed the urinary nitrate-to-nitrite molar ratio, i.e., UNOxR, as a measure of renal CA-dependent excretion of nitrite. Based on results from clinical and experimental animal studies, here, we report on a first evaluation of UNOxR. We determined UNOxR values in preterm neonates, healthy children, and adults, in children suffering from type 1 diabetes mellitus (T1DM) or Duchenne muscular dystrophy (DMD), in elderly subjects suffering from chronic rheumatic diseases, type 2 diabetes mellitus (T2DM), coronary artery disease (CAD), or peripheral arterial occlusive disease (PAOD). We also determined UNOxR values in healthy young men who ingested isosorbide dinitrate (ISDN), pentaerythrityl tetranitrate (PETN), or inorganic nitrate. In addition, we tested the utility of UNOxR in two animal models, i.e., the LEW.1AR1-iddm rat, an animal model of human T1DM, and the APOE*3-Leiden.CETP mice, a model of human dyslipidemia. Mean UNOxR values were lower in adult patients with rheumatic diseases (187) and in T2DM patients of the DALI study (74) as compared to healthy elderly adults (660) and healthy young men (1500). The intra- and inter-variabilities of UNOxR were of the order of 50% in young and elderly healthy subjects. UNOxR values were lower in black compared to white boys (314 vs. 483, P = 0.007), which is in line with reported lower NO bioavailability in black ethnicity. Mean UNOxR values were lower in DMD (424) compared to healthy (730) children, but they were higher in T1DM children (1192). ISDN (3 × 30 mg) decreased stronger UNOxR compared to PETN (3 × 80 mg) after 1 day (P = 0.046) and after 5 days (P = 0.0016) of oral administration of therapeutically equivalent doses. In healthy young men who ingested NaNO3 (0.1 mmol/kg/d), UNOxR was higher than in those who ingested the same dose of NaCl (1709 vs. 369). In LEW.1AR1-iddm rats, mean UNOxR values were lower than in healthy rats (198 vs. 308) and comparable to those in APOE*3-Leiden.CETP mice (151).

A novel Dock8 gene mutation confers diabetogenic susceptibility in the LEW.1AR1/Ztm-iddm rat, an animal model of human type 1 diabetes.

AIMS/HYPOTHESIS: The LEW.1AR1-iddm rat, an animal model of human type 1 diabetes, arose through a spontaneous mutation within the inbred strain LEW.1AR1. A susceptibility locus (Iddm8) on rat chromosome 1 (RNO1) has been identified previously, which is accompanied by autoimmune diabetes and the additional phenotype of a variable CD3(+) T cell frequency. METHODS: In the present study we characterised the Iddm8 region on RNO1 in backcross strains using the genetically divergent Brown Norway (BN) and Paris (PAR) rats. Candidate genes of the Iddm8 region were sequenced for mutation analysis. RESULTS: The Iddm8 region could be subdivided by single nucleotide polymorphism (SNP) analyses. In the first region, a mutation in exon 44 of the Dock8 gene was identified resulting in an amino acid exchange in the protein from glutamine to glutamate. This exchange is unique for the LEW.1AR1-iddm rat. In the second region, a SNP was detected in exon 11 of the Vwa2 gene with an exchange from arginine to tryptophan. This SNP is also present in other rat strains. CONCLUSIONS/INTERPRETATION: The Dock8 mutation gave rise to a new type 1 diabetes rat model with very close similarity to type 1 diabetes in humans, providing a deepened insight into the impact of genes involved in diabetes development.

Islet infiltration, cytokine expression and beta cell death in the NOD mouse, BB rat, Komeda rat, LEW.1AR1-iddm rat and humans with type 1 diabetes.

AIMS/HYPOTHESIS: Research on the pathogenesis of type 1 diabetes relies heavily on good animal models. The aim of this work was to study the translational value of animal models of type 1 diabetes to the human situation. METHODS: We compared the four major animal models of spontaneous type 1 diabetes, namely the NOD mouse, BioBreeding (BB) rat, Komeda rat and LEW.1AR1-iddm rat, by examining the immunohistochemistry and in situ RT-PCR of immune cell infiltrate and cytokine pattern in pancreatic islets, and by comparing findings with human data. RESULTS: After type 1 diabetes manifestation CD8(+) T cells, CD68(+) macrophages and CD4(+) T cells were observed as the main immune cell types with declining frequency, in infiltrated islets of all diabetic pancreases. IL-1ß and TNF-α were the main proinflammatory cytokines in the immune cell infiltrate in NOD mice, BB rats and LEW.1AR1-iddm rats, as well as in humans. The Komeda rat was the exception, with IFN-γ and TNF-α being the main cytokines. In addition, IL-17 and IL-6 and the anti-inflammatory cytokines IL-4, IL-10 and IL-13 were found in some infiltrating immune cells. Apoptotic as well as proliferating beta cells were observed in infiltrated islets. In healthy pancreases no proinflammatory cytokine expression was observed. CONCLUSIONS/INTERPRETATION: With the exception of the Komeda rat, the animal models mirror very well the situation in humans with type 1 diabetes. Thus animal models of type 1 diabetes can provide meaningful information on the disease processes in the pancreas of patients with type 1 diabetes.

Rat Models of Human Type 1 Diabetes.

Rat models of human type 1 diabetes have been shown to be of great importance for the elucidation of the mechanisms underlying the development of autoimmune diabetes. The three major well-established spontaneous rat models are the BioBreeding (BB) diabetes-prone rat, the Komeda diabetes-prone (KDP) rat, and the IDDM (LEW.1AR1-iddm) rat. Their distinctive features are described with special reference to their pathology, immunology, and genetics and compared with the situation in patients with type 1 diabetes mellitus. For all three established rat models, a distinctive genetic mutation has been identified that is responsible for the manifestation of the diabetic syndrome in these rat strains.

Translation of curative therapy concepts with T cell and cytokine antibody combinations for type 1 diabetes reversal in the IDDM rat.

Proinflammatory cytokines released from the pancreatic islet immune cell infiltrate in type 1 diabetes (T1D) cause insulinopenia as a result of severe beta cell loss due to apoptosis. Diabetes prevention strategies targeting different cytokines with antibodies in combination with a T cell antibody, anti-TCR, have been assessed for therapy success in the LEW.1AR1-iddm (IDDM) rat, an animal model of human T1D. Immediately after diabetes manifestation, antibody combination therapies were initiated over 5 days with anti-TNF-α (tumour necrosis factor), anti-IL-1ß (interleukin), or anti-IFN-γ (interferon) together with anti-TCR for the reversal of the diabetic metabolic state in the IDDM rat. Anti-TCR alone showed only a very limited therapy success with respect to a reduction of immune cell infiltration and beta cell mass regeneration. Anti-TCR combinations with anti-IL-1ß or anti-IFN-γ were also not able to abolish the increased beta cell apoptosis rate and the activated immune cell infiltrate leading to a permanent beta cell loss. In contrast, all anti-TCR combinations with anti-TNF-α provided sustained therapy success over 60 to 360 days. The triple combination of anti-TCR with anti-TNF-α plus anti-IL-1ß was most effective in regaining sustained normoglycaemia with an intact islet structure in a completely infiltration-free pancreas and with a normal beta cell mass. Besides the triple combination, the double antibody combination of anti-TCR with anti-TNF-α proved to be the most suited therapy for reversal of the T1D metabolic state due to effective beta cell regeneration in an infiltration free pancreas. KEY MESSAGES: Anti-TCR is a cornerstone in combination therapy for autoimmune diabetes reversal. The combination of anti-TCR with anti-TNF-α was most effective in reversing islet immune cell infiltration. Anti-TCR combined with anti-IL-1ß was not effective in this respect. The combination of anti-TCR with anti-TNF-α showed a sustained effect over 1 year.

Changes in immune cell frequencies in primary and secondary lymphatic organs of LEW.1AR1-iddm rats, a model of human type 1 diabetes compared to other MHC congenic LEW inbred strains.

The LEW.1AR1-iddm rat is an animal model of human type 1 diabetes, which arose through a spontaneous mutation in the Dock8 gene within the MHC congenic background strain LEW.1AR1. This mutation not only mediates diabetes development but also leads to a variable T cell frequency in peripheral blood. In this study, the immune cell frequencies of primary and secondary lymphatic organs of LEW.1AR1-iddm rats were analysed at days 40 and 60 and compared to other MHC congenic LEW rat strains. In LEW.1AR1-iddm rats, the secondary lymphatic organs such as lymph nodes and spleen showed a reduced, around 15% in comparison to all other strains, but very variable T cell frequency, mirroring the fluctuating T cell content in blood. On the other hand, the frequency of B cells was increased by 10% in the lymph nodes and by 5% in the spleen. Thus, the decreasing number of T cells in blood could not be caused by an increase of T cells in secondary lymphatic organs. The frequency of single- or double-positive T cells in the thymus was unaffected. The T cell frequencies in the other analysed strains were more stable and mostly higher in all secondary lymphatic organs. Obviously, the Dock8 mutation leads to variabilities of T cell frequencies in blood as well as in secondary lymphatic organs. In conclusion, the Dock8 mutation was responsible for changed immune cell frequencies in different compartments and together with the RT1B/Du haplotype causing immune imbalances and development of autoimmune diabetes.

Pattern of genes influenced by conditional expression of the transcription factors HNF6, HNF4alpha and HNF1beta in a pancreatic beta-cell line.

Using the rat insulinoma cell line INS-1 we generated beta-cell clones that are most efficient for gene transfer, as they contain an FRT site for Flp recombinase-mediated, site-directed integration of a single copy transgene. Therefore, the gene-of-interest can be introduced by DNA transfection without the need to select individual cell clones. Additionally, the clones contain the tetracycline repressor allowing tetracycline induction of the transgene. By oligonucleotide microarray we define the beta-cell specific phenotype of the Flp-In T-REx cell clones. Using a clone expressing the HNF6, HNF4alpha and HNF1beta transcription factors at a limited level, we introduced the expression vectors encoding these factors. We show efficient tetracycline induction of these transcription factors by western blots and immunocytochemistry. Microarrays reveal that these three factors affect a similar number of genes with only few genes regulated in common. Statistical analysis reveals that the three transcription factors affect genes categorized to different biological processes. Furthermore, we document the usefulness of these Flp-In T-REx cells for the functional analysis of mutated HNF1beta transcription factors found in human MODY5 patients. We show that the expression of the mutant P328L329del and A263insGG affects only very few transcripts and these are predominantly distinct from those induced by wild-type HNF1beta.

TNF-α Antibody Therapy in Combination With the T-Cell-Specific Antibody Anti-TCR Reverses the Diabetic Metabolic State in the LEW.1AR1-iddm Rat.

Anti-tumor necrosis factor-α (TNF-α) therapy (5 mg/kg body weight), alone or combined with the T-cell-specific antibody anti-T-cell receptor (TCR) (0.5 mg/kg body weight), was performed over 5 days immediately after disease manifestation to reverse the diabetic metabolic state in the LEW.1AR1-iddm rat, an animal model of human type 1 diabetes. Only combination therapy starting at blood glucose concentrations below 15 mmol/L restored normoglycemia and normalized C-peptide. Increased ß-cell proliferation and reduced apoptosis led to a restoration of ß-cell mass along with an immune cell infiltration-free pancreas 60 days after the end of therapy. This combination of two antibodies, anti-TCR/CD3, as a cornerstone compound in anti-T-cell therapy, and anti-TNF-α, as the most prominent and effective therapeutic antibody in suppressing TNF-α action in many autoimmune diseases, was able to reverse the diabetic metabolic state. With increasing blood glucose concentrations during the disease progression, however, the proapoptotic pressure on the residual ß-cell mass increased, ultimately reaching a point where the reservoir of the surviving ß-cells was insufficient to allow a restoration of normal ß-cell mass through regeneration. The present results may open a therapeutic window for reversal of diabetic hyperglycemia in patients, worthwhile of being tested in clinical trials.

Anti-TCR therapy combined with fingolimod for reversal of diabetic hyperglycemia by ß cell regeneration in the LEW.1AR1-iddm rat model of type 1 diabetes.

UNLABELLED: The therapeutic capacity of an antibody directed against the T cell receptor (anti-TCR) of the TCR/CD3 complex alone or in combination with fingolimod (FTY720) to reverse the diabetic metabolic state through suppression of autoimmunity and stimulation of ß cell regeneration was analyzed in the LEW.1AR1-iddm (IDDM) rat, an animal model of human type 1 diabetes. Animals were treated with anti-TCR (0.5 mg/kg body weight for 5 days) monotherapy or in combination with fingolimod (1 mg/kg body weight for 40 days). Metabolic changes and ß cell morphology were analyzed before, immediately after, and 60 days after end of therapy. Both therapies were started early after disease manifestation and led to normoglycemia in parallel with an increase of the C-peptide concentration. Combination therapy increased the ß cell mass reaching a range of normoglycemic controls, decreased the apoptosis rate fivefold, and increased the proliferation rate threefold. Additionally, at 60 days after therapy, islets were virtually free of T cells, macrophages, and cytokine expression. In contrast, after anti-TCR monotherapy, ß cell mass remained low with an activated immune cell infiltrate. A concomitant fivefold increased ß cell apoptosis rate resulted in a complete loss of ß cells. Only combination therapy yielded sustained normoglycemia with full reversal of islet infiltration and restoration of pancreatic ß cell mass. KEY MESSAGE: Combination therapy of anti-TCR and fingolimod was effective in the reversal of T1D. Combination therapy increased the pancreatic ß cell mass to normoglycemic control levels. Combination therapy leads to a full reversal of pancreatic islet infiltration. Anti-TCR monotherapy did not abolish islet infiltration. Combination therapy was successful only immediately after diabetes manifestation.

A variable CD3⁺ T-cell frequency in peripheral blood lymphocytes associated with type 1 diabetes mellitus development in the LEW.1AR1-iddm rat.

PURPOSE: The LEW.1AR1-iddm rat is an animal model of human type 1 diabetes mellitus (T1DM), which arose through a spontaneous mutation within the MHC-congenic inbred strain LEW.1AR1 (RT1(r²)). In contrast to the diabetes-resistant LEW.1AR1 background strain in LEW.1AR1-iddm rats a highly variable T-cell frequency could be observed in peripheral blood lymphocytes (PBLs). METHODS: In this study we therefore characterised the T-cell repertoire within the PBLs of the two strains by flow cytometry analysis and identified the CD3⁺ T-cell phenotype and its possible linkage to diabetes susceptibility. To map loci conferring susceptibility to variable CD3⁺ T-cell frequency, backcross strains (N2) were generated with the genetically divergent BN and PAR rats for microsatellite analysis. RESULTS: The LEW.1AR1-iddm rat strain was characterised by a higher variability of CD3⁺ T-cells in PBLs along with a slightly decreased mean value compared to the LEW.1AR1 background strain. The reason for this reduction was a decrease in the CD4⁺ T-cell count while the CD8⁺ T-cell proportion remained unchanged. However, both T-cell subpopulations showed a high variability. This resulted in a lower CD4⁺/CD8⁺ T-cell ratio than in LEW.1AR1 rats. Like LEW.1AR1-iddm rats all animals of the backcross populations, N2 BN and N2 PAR rats, also showed large variations of the CD3⁺ T-cell frequency. The phenotype of variable CD3⁺ T-cell frequency mapped to the telomeric region of chromosome 1 (RNO1), which is identical with the already known Iddm8 diabetes susceptibility region. The data indicate that a variable CD3⁺ T-cell frequency in PBLs is genetically linked to diabetes susceptibility in the LEW.1AR1-iddm rat. CONCLUSION: The T-cell variability in PBLs could be related to the previously reported imbalance between regulatory and effector T-cell populations which results in beta-cell autoimmunity. Since similar T-cell phenotypes have also been described in human T1DM the identification of the functional role of the observed variable CD3⁺ T-cell frequency may help to understand the mechanisms of autoimmunity in T1DM.

Diabetes prevention by immunomodulatory FTY720 treatment in the LEW.1AR1-iddm rat despite immune cell activation.

The prevention of diabetes by the immunomodulatory agent FTY720 (fingolimod) was studied in the LEW.1AR1-iddm (IDDM) rat, an animal model of human type 1 diabetes. Immune cell subtypes and cytokine profiles in pancreatic islets, secondary lymphoid tissue, and serum were analyzed for signs of immune cell activation. Animals were treated with FTY720 (1 mg/kg body weight) for 40 d starting on d 50 of life. Changes in gene and protein expression of cytokines, CD8 markers, monocyte chemoattractant protein-1, inducible NO synthase, and caspase 3 were evaluated. Treatment with FTY720 prevented diabetes manifestation and islet infiltration around d 60 of life, the usual time of spontaneous diabetes development. On d 120, 30 d after the end of FTY720 therapy, diabetes prevention persisted. However, six of 12 treated animals showed increased gene expression of IL-1beta, TNF-alpha, and CD8 markers in pancreas-draining lymph nodes, indicating immune cell activation. In parallel, serum concentrations of these proinflammatory cytokines were increased. These six animals also showed macrophage infiltration without proinflammatory cytokine expression in a small minority (2-3%) of islets. Interestingly, regulatory T lymphocytes were significantly increased in the efferent vessels of the pancreas-draining lymph nodes only in animals without signs of immune cell activation but not in the rats with immune cell activation. This provides an indication for a lack of protective capacity in the animals with activated immune cells. Thus, FTY720 treatment prevented the manifestation of diabetes by promoting the retention of activated immune cells in the lymph nodes, thereby avoiding islet infiltration and beta-cell destruction by proinflammatory cytokines.

The mutation of the LEW.1AR1-iddm rat maps to the telomeric end of rat chromosome 1.

The LEW.1AR1-iddm rat is an animal model of human type 1 diabetes mellitus (T1DM) with an autosomal recessive mode of inheritance. T1DM susceptibility loci could be localized on chromosome (RNO) 20 in the major histocompatibility complex region (Iddm1) and on RNO1 (Iddm8, Iddm9) in a BN backcross cohort. In this study the impact of the different susceptibility regions on diabetes development was investigated in a backcross population of the diabetes-resistant PAR strain. A cohort of 130 [(PAR x LEW.1AR1-iddm) x LEW.1AR1-iddm] N2 rats was monitored for blood glucose and analyzed by linkage analysis. Sixteen percent of the PAR backcross animals developed T1DM. Genetic analysis revealed significant linkage to T1DM in the MHC region on RNO20p12. In contrast to the linkage analysis of the BN backcross cohort, only one susceptibility locus for T1DM could be identified on RNO1. This susceptibility region on RNO1 mapped to the telomeric end corresponding to Iddm8. Eighty-nine percent of diabetic PAR backcross animals were homozygous for Iddm8. The Iddm9 diabetes susceptibility region showed no linkage to diabetes in the PAR backcross cohort. The data of this study provide evidence that the mutation leading to T1DM in the LEW.1AR1-iddm rat is located at the telomeric end of RNO1 corresponding to Iddm8.

Tissue-specific transcription factor HNF4alpha inhibits cell proliferation and induces apoptosis in the pancreatic INS-1 beta-cell line.

Hepatocyte nuclear factor 4alpha (HNF4alpha) is a tissue-specific transcription factor expressed in many cell types, including pancreatic beta-cells. Mutations in the HNF4alpha gene in humans give rise to maturity-onset diabetes of the young (MODY1) characterized by defective insulin secretion by beta-cells. To elucidate the mechanism underlying this disease, we introduced the splice form HNF4alpha2 or HNF4alpha8 into the rat beta-cell line INS-1. Upon tetracycline-induced expression, both HNF4alpha isoforms caused distinct changes in cell morphology and a massive loss of cell numbers that was correlated with reduced proliferation and induced apoptosis. This differential activity was reflected in oligonucleotide microarray analysis that identified more genes affected by HNF4alpha2 compared to HNF4alpha8, and suggests that both isoforms regulate largely the same set of genes, with HNF4alpha2 being a stronger transactivator. We verified the induction of selected transcripts by real-time RT-PCR, including KAI1 and AIF, both known to have apoptotic potential. By establishing cell lines with inducible expression of these target genes, we deduce that both factors are insufficient to induce apoptosis. We propose that the anti-proliferative and apoptotic properties of HNF4alpha may be an essential feature impaired in MODY1 and possibly also in type 2 diabetes.

Kinetics of CD8+ effector T cell responses and induced CD4+ regulatory T cell responses during Friend retrovirus infection.

Cytolytic CD8+ T cells are critical for the control of acute Friend virus (FV) infection yet they fail to completely eliminate the virus during chronic infection because they are functionally impaired by regulatory T cells (Treg). We performed a kinetic analysis of T cell responses during FV infection to determine when dysfunction of CD8+ T cells and suppressive activity of CD4+ regulatory T cells develops. At 1 week post infection, virus-specific CD8+ T cells with effector phenotype and cytolytic potential expanded. Peak expansion was found at 12 days post infection, correlating with peak viral loads. After 2 weeks when viral loads dropped, numbers of activated CD8+ T cells started to decline. However, a population of virus-specific CD8+ T cells with effector phenotype was still detectable subsequently, but these cells had lost their ability to produce granzymes and to degranulate cytotoxic molecules. Contemporaneous with the development of CD8+ T cell dysfunction, different CD4+ T cell populations expressing cell surface markers for Treg and the Treg-associated transcription factor Foxp3 expanded. Transfer as well as depletion experiments indicated that regulatory CD4+ cells developed during the second week of FV infection and subsequently suppressed CD8+ T cell functions, which was associated with impaired virus clearance.

Effective treatment of retrovirus-induced suppression of antibody responses with CpG oligodeoxynucleotides.

Most retroviruses induce severe immunosuppression during acute infection. We have used the Friend retrovirus mouse model to demonstrate that immunostimulatory B-type CpG oligodeoxynucleotides (ODN) have a protective effect against retrovirus-induced suppression of antibody responses to potent B-cell antigens. CD8+ T cells were critical for effective treatment with CpG-ODN, since in vivo depletion of these cells from treated mice impaired protection from retrovirus-induced immunosuppression. Protection also required IFN-gamma, as neutralization of this cytokine abolished the therapeutic effect of CpG-ODN. These findings may have implications for the treatment of immunosuppressive virus infections.