Arylphthalide Delays Diabetic Retinopathy via Immunomodulating the Early Inflammatory Response in an Animal Model of Type 1 Diabetes Mellitus.

Diabetic retinopathy (DR) is one of the most prevalent secondary complications associated with diabetes. Specifically, Type 1 Diabetes Mellitus (T1D) has an immune component that may determine the evolution of DR by compromising the immune response of the retina, which is mediated by microglia. In the early stages of DR, the permeabilization of the blood-retinal barrier allows immune cells from the peripheral system to interact with the retinal immune system. The use of new bioactive molecules, such as 3-(2,4-dihydroxyphenyl)phthalide (M9), with powerful anti-inflammatory activity, might represent an advance in the treatment of diseases like DR by targeting the immune systems responsible for its onset and progression. Our research aimed to investigate the molecular mechanisms involved in the interaction of specific cells of the innate immune system during the progression of DR and the reduction in inflammatory processes contributing to the pathology. In vitro studies were conducted exposing Bv.2 microglial and Raw264.7 macrophage cells to proinflammatory stimuli for 24 h, in the presence or absence of M9. Ex vivo and in vivo approaches were performed in BB rats, an animal model for T1D. Retinal explants from BB rats were cultured with M9. Retinas from BB rats treated for 15 days with M9 via intraperitoneal injection were analyzed to determine survival, cellular signaling, and inflammatory markers using qPCR, Western blot, or immunofluorescence approaches. Retinal structure images were acquired via Spectral-Domain-Optical Coherence Tomography (SD-OCT). Our results show that the treatment with M9 significantly reduces inflammatory processes in in vitro, ex vivo, and in vivo models of DR. M9 works by inhibiting the proinflammatory responses during DR progression mainly affecting immune cell responses. It also induces an anti-inflammatory response, primarily mediated by microglial cells, leading to the synthesis of Arginase-1 and Hemeoxygenase-1(HO-1). Ultimately, in vivo administration of M9 preserves the retinal integrity from the degeneration associated with DR progression. Our findings demonstrate a specific interaction between both retinal and systemic immune cells in the progression of DR, with a differential response to treatment, mainly driven by microglia in the anti-inflammatory action. In vivo treatment with M9 induces a switch in immune cell phenotypes and functions that contributes to delaying the DR progression, positioning microglial cells as a new and specific therapeutic target in DR.

Expression analysis of microRNAs and their target genes during experimental diabetic renal lesions in rats administered with ginsenoside Rb1 and trigonelline.

Purpose: To appraise the curative effect of ginsenoside Rb1 and trigonelline in diabetic nephropathy and to analyze the expression analysis of microRNAs and their target genes during experimental diabetic renal lesions in rats. Methods: Wistar rats were made diabetic by intraperitoneal injection of 55 mg/kg streptozotocin. According to their fasting blood glucose values and initial body weight, diabetic rats were assigned to specific groups and treated as follows: DN group (tap water, n = 10), A group (ginsenoside Rb1, 40 mg/kg, n = 10), B group (trigonelline, 20 mg/kg, n = 10) and the C group (ginsenoside Rb1 and trigonelline, 60 mg/kg, m(ginsenoside Rb1) : m (trigonelline) = 2:1, n = 10). The control group was treated with tap water (n = 10). All rats were gavaged with drugs or tap water once daily for 12 weeks. Results: Renal dysfunction, oxidative stress, and pathological alteration were significantly alleviated by a combination of ginsenoside Rb1 and trigonellin (C group). Some miRNAs were expressed differentially in Con, DN, A and C groups. Results of immunohistochemistry and western blotting showed that Wnt and ß-catenin were expressed differentially in Con, DN, and C groups. Conclusion: Ginsenoside Rb1 and trigonelline could prevent the development of diabetic renal lesions by regulating the expression of miR-3550 and further associating with the Wnt/ß-catenin signaling.

Early deficits in insulin secretion, beta cell mass and islet blood perfusion precede onset of autoimmune type 1 diabetes in BioBreeding rats.

AIMS/HYPOTHESIS: Genetic studies show coupling of genes affecting beta cell function to type 1 diabetes, but hitherto no studies on whether beta cell dysfunction could precede insulitis and clinical onset of type 1 diabetes are available. METHODS: We used 40-day-old BioBreeding (BB) DRLyp/Lyp rats (a model of spontaneous autoimmune type 1 diabetes) and diabetes-resistant DRLyp/+ and DR+/+ littermates (controls) to investigate beta cell function in vivo, and insulin and glucagon secretion in vitro. Beta cell mass was assessed by optical projection tomography (OPT) and morphometry. Additionally, measurements of intra-islet blood flow were performed using microsphere injections. We also assessed immune cell infiltration, cytokine expression in islets (by immunohistochemistry and qPCR), as well as islet Glut2 expression and ATP/ADP ratio to determine effects on glucose uptake and metabolism in beta cells. RESULTS: DRLyp/Lyp rats were normoglycaemic and without traces of immune cell infiltrates. However, IVGTTs revealed a significant decrease in the acute insulin response to glucose compared with control rats (1685.3 ± 121.3 vs 633.3 ± 148.7; p < 0.0001). In agreement, insulin secretion was severely perturbed in isolated islets, and both first- and second-phase insulin release were lowered compared with control rats, while glucagon secretion was similar in both groups. Interestingly, after 5-7 days of culture of islets from DRLyp/Lyp rats in normal media, glucose-stimulated insulin secretion (GSIS) was improved; although, a significant decrease in GSIS was still evident compared with islets from control rats at this time (7393.9 ± 1593.7 vs 4416.8 ± 1230.5 pg islet-1 h-1; p < 0.0001). Compared with controls, OPT of whole pancreas from DRLyp/Lyp rats revealed significant reductions in medium (4.1 × 109 ± 9.5 × 107 vs 3.8 × 109 ± 5.8 × 107 µm3; p = 0.044) and small sized islets (1.6 × 109 ± 5.1 × 107 vs 1.4 × 109 ± 4.5 × 107 µm3; p = 0.035). Finally, we found lower intra-islet blood perfusion in vivo (113.1 ± 16.8 vs 76.9 ± 11.8 µl min-1 [g pancreas]-1; p = 0.023) and alterations in the beta cell ATP/ADP ratio in DRLyp/Lyp rats vs control rats. CONCLUSIONS/INTERPRETATION: The present study identifies a deterioration of beta cell function and mass, and intra-islet blood flow that precedes insulitis and diabetes development in animals prone to autoimmune type 1 diabetes. These underlying changes in islet function may be previously unrecognised factors of importance in type 1 diabetes development.

Increased Activation of the TRESK K+ Mediates Vago-Vagal Reflex Malfunction in Diabetic Rats.

BACKGROUND & AIMS: Patients with diabetes have defects in the vagal afferent pathway that result in abnormal gastrointestinal function. We investigated whether selective increased activation of the 2-pore domain potassium channel TRESK (2-pore-domain weak inward-rectifying potassium channel-related spinal cord potassium channel) contributes to nodose ganglia (NG) malfunction, disrupting gastrointestinal function in diabetic rats. METHODS: We conducted whole-cell current-clamp and single-unit recordings in NG neurons from diabetes-prone BioBreeding/Worcester rats and streptozotocin-induced diabetic (STZ-D) rats and compared them with control rats. NG neurons in rats or cultured NG neurons were exposed to pharmacologic antagonists and/or transfected with short hairpin or small interfering RNAs that reduced expression of TRESK. We then made electrophysiologic recordings and studied gastrointestinal functions. RESULTS: We observed reduced input resistance, hyperpolarized membrane potential, and increased current threshold to elicit action potentiation in NG neurons of STZ-D rats compared with controls. NG neuron excitability was similarly altered in diabetes-prone rats. In vivo single-unit NG neuronal discharges in response to 30 and 60 pmol cholecystokinin octapeptide were significantly lower in STZ-D rats compared with controls. Reducing expression of the TRESK K+ channel restored NG excitability in vitro and in vivo, as well as cholecystokinin 8-stimulated secretion of pancreatic enzymes and secretin-induced gastrointestinal motility, which are mediated by vago-vagal reflexes. These abnormalities resulted from increased intracellular Ca2+ in the NG, activating calcineurin, which, in turn, bound to an nuclear factor of activated T cell-like docking site on the TRESK protein, resulting in neuronal membrane hyperpolarization. CONCLUSIONS: In 2 rate models of diabetes, we found that activation of the TRESK K+ channel reduced NG excitability and disrupted gastrointestinal functions.

Ghrelin mitigates ß-cell mass loss during insulitis in an animal model of autoimmune diabetes mellitus, the BioBreeding/Worcester rat.

BACKGROUND: Ghrelin is a peptide hormone with pleiotropic effects. It stimulates cell proliferation and inhibits apoptosis-mediated cell death. It prevents diabetes mellitus in several models of chemical, surgical and biological toxic insults to pancreas in both in vivo and in vitro models and promotes glucose-stimulated insulin secretion under cytotoxic conditions. It has not yet been tested in vivo in an autoimmune model of diabetes with a persistent insult to the ß-cell. Given the immunomodulating effects of ghrelin and its trophic effects on ß-cells, we hypothesized that ghrelin treatment during the early stages of insulitis would delay diabetes onset. METHODS: BioBreeding/Worcester male rats received ghrelin (10 ng/kg/day) before insulitis development. Glucose metabolism was characterized by glucose and insulin tolerance tests. ß-cell mass, islet area, islet number, ß-cell clusters, proliferation and apoptosis and degree of insulitis were analysed by histomorphometry. A Kaplan-Meier survival curve was plotted and analysed applying the log-rank (Mantel-Cox) test. RESULTS: Ghrelin treatment significantly reduced the probability of developing diabetes in our model (p < 0.0001). It decreased islet infiltration and partially prevented ß-cell mass loss, enabling the maintenance of ß-cell neogenesis and proliferation rates. Furthermore, ghrelin treatment did not induce any metabolic perturbations. CONCLUSIONS: These findings support the hypothesis that ghrelin delays the development of autoimmune diabetes by attenuating insulitis and supporting ß-cell mass. GENERAL SIGNIFICANCE: Ghrelin promotes ß-cell viability and function through diverse mechanisms that may have significant implications for diabetes prevention, therapy and also transplant success of both islets and complete pancreas. Copyright © 2016 John Wiley & Sons, Ltd.

The normoglycaemic biobreeding rat: a spontaneous model for impaired gastric accommodation.

OBJECTIVE: Impaired gastric accommodation is reported in patients with functional dyspepsia (FD). Previous findings in postinfectious patients with FD suggest that low-grade inflammation and dysfunction of nitrergic nerves play a role in impaired accommodation. To date, spontaneous animal models to study the relationship between these changes are lacking. We hypothesise that the normoglycaemic BioBreeding diabetes-prone (BB-DP) rat provides an animal model of inflammation-induced impaired gastric motor function. DESIGN: Control diabetes-resistant biobreeding, normoglycaemic and hyperglycaemic BB-DP rats were sacrificed at the age of 30, 70 and 220 days and gastric fundus tissue was harvested to study nitrergic motor control, inflammation and expression of neuronal isoform of nitric oxide synthase (nNOS) and inducible isoform of nitric oxide synthase (iNOS). Nutrient-induced changes in intragastric pressure (IGP) were measured in normoglycaemic BB-DP rats to study accommodation. RESULTS: No differences in nitrergic function and inflammation were observed between BB-DP and control rats at 30 days. The nitrergic component of the fundic muscle relaxation was reduced in BB-DP rats of 70 and 220 days. This was accompanied by a significant loss of nNOS proteins. IGP significantly increased during nutrient infusion in BB-DP rats of 220 days, indicating impaired accommodation. Infiltration of polymorphonuclear cells, increased myeloperoxidase activity and increased expression of iNOS was observed in the fundic mucosa and muscularis propria of 70-day-old and 220-day-old BB-DP rats. CONCLUSIONS: BB-DP rats of 220 days display altered fundic motor control and impaired accommodation, which is least partially explained by loss of nitrergic function. This may be related to inflammatory changes in the neuromuscular layer, suggesting that normoglycaemic BB-DP rats provide a spontaneous model for inflammation-induced impaired gastric accommodation.

The importance of the Non Obese Diabetic (NOD) mouse model in autoimmune diabetes.

Type 1 Diabetes (T1D) is an autoimmune disease characterized by the pancreatic infiltration of immune cells resulting in T cell-mediated destruction of the insulin-producing beta cells. The successes of the Non-Obese Diabetic (NOD) mouse model have come in multiple forms including identifying key genetic and environmental risk factors e.g. Idd loci and effects of microorganisms including the gut microbiota, respectively, and how they may contribute to disease susceptibility and pathogenesis. Furthermore, the NOD model also provides insights into the roles of the innate immune cells as well as the B cells in contributing to the T cell-mediated disease. Unlike many autoimmune disease models, the NOD mouse develops spontaneous disease and has many similarities to human T1D. Through exploiting these similarities many targets have been identified for immune-intervention strategies. Although many of these immunotherapies did not have a significant impact on human T1D, they have been shown to be effective in the NOD mouse in early stage disease, which is not equivalent to trials in newly-diagnosed patients with diabetes. However, the continued development of humanized NOD mice would enable further clinical developments, bringing T1D research to a new translational level. Therefore, it is the aim of this review to discuss the importance of the NOD model in identifying the roles of the innate immune system and the interaction with the gut microbiota in modifying diabetes susceptibility. In addition, the role of the B cells will also be discussed with new insights gained through B cell depletion experiments and the impact on translational developments. Finally, this review will also discuss the future of the NOD mouse and the development of humanized NOD mice, providing novel insights into human T1D.

Iddm30 controls pancreatic expression of Ccl11 (Eotaxin) and the Th1/Th2 balance within the insulitic lesions.

The autoimmune diabetic syndrome of the BioBreeding diabetes-prone (BBDP) rat is a polygenic disease that resembles in many aspects human type 1 diabetes (T1D). A successful approach to gain insight into the mechanisms underlying genetic associations in autoimmune diseases has been to identify and map disease-related subphenotypes that are under simpler genetic control than the full-blown disease. In this study, we focused on the ß cell overexpression of Ccl11 (Eotaxin), previously postulated to be diabetogenic in BBDR rats, a BBDP-related strain. We tested the hypothesis that this trait is genetically determined and contributes to the regulation of diabetes in BBDP rats. Similar to the BBDR strain, we observed a time-dependent, insulitis-independent pancreatic upregulation of Ccl11 in BBDP rats when compared with T1D-resistant ACI.1u.lyp animals. Through linkage analysis of a cross-intercross of these two parental strains, this trait was mapped to a region on chromosome 12 that overlaps Iddm30. Linkage results were confirmed by phenotypic assessment of a novel inbred BBDP.ACI-Iddm30 congenic line. As expected, the Iddm30 BBDP allele is associated with a significantly higher pancreatic expression of Ccl11; however, the same allele confers resistance to T1D. Analysis of islet-infiltrating T cells in Iddm30 congenic BBDP animals revealed that overexpression of pancreatic Ccl11, a prototypical Th2 chemokine, is associated with an enrichment in Th2 CD4+ T cells within the insulitic lesions. These results indicate that, in the BBDP rat, Iddm30 controls T1D susceptibility through both the regulation of Ccl11 expression in ß cells and the subsequent Th1/Th2 balance within islet-infiltrating T lymphocytes.

Gut immune deficits in LEW.1AR1-iddm rats partially overcome by feeding a diabetes-protective diet.

The gut immune system and its modification by diet have been implicated in the pathogenesis of type 1 diabetes (T1D). Therefore, we investigated gut immune status in non-diabetes-prone LEW.1AR1 and diabetes-prone LEW.1AR1-iddm rats and evaluated the effect of a low antigen, hydrolysed casein (HC)-based diet on gut immunity and T1D. Rats were weaned onto a cereal-based or HC-based diet and monitored for T1D. Strain and dietary effects on immune homeostasis were assessed in non-diabetic rats (50-60 days old) and rats with recent-onset diabetes using flow cytometry and immunohistochemistry. Immune gene expression was analysed in mesenteric lymph nodes (MLN) and jejunum using quantitative RT-PCR and PCR arrays. T1D was prevented in LEW.1AR1-iddm rats by feeding an HC diet. Diabetic LEW.1AR1-iddm rats had fewer lymphoid tissue T cells compared with LEW.1AR1 rats. The percentage of CD4(+)  Foxp3(+) regulatory T (Treg) cells was decreased in pancreatic lymph nodes (PLN) of diabetic rats. The jejunum of 50-day LEW.1AR1-iddm rats contained fewer CD3(+) T cells, CD163(+) M2 macrophages and Foxp3(+) Treg cells. Ifng expression was increased in MLN and Foxp3 expression was decreased in the jejunum of LEW.1AR1-iddm rats; Ifng/Il4 was decreased in jejunum of LEW.1AR1-iddm rats fed HC. PCR arrays revealed decreased expression of M2-associated macrophage factors in 50-day LEW.1AR1-iddm rats. Wheat peptides stimulated T-cell proliferation and activation in MLN and PLN cells from diabetic LEW.1AR1-iddm rats. LEW.1AR1-iddm rats displayed gut immune cell deficits and decreased immunoregulatory capacity, which were partially corrected in animals fed a low antigen, protective HC diet consistent with other models of T1D.

Genetic dissection of Iddm26 in the spontaneously diabetic BBDP rat.

The 40 Mb T1D susceptibility locus Iddm26 was mapped to chromosome 2 through linkage analysis of a conditioned cross-intercross between the diabetes-prone BBDP and the diabetes-resistant ACI.BBDP-Iddm1,Iddm2 (ACI.1u.Lyp). It is flanked by Iddm32 and Iddm33, which control the kinetics of disease progression. To fine-map Iddm26 and characterize immune phenotypes controlled by this locus, several congenic sublines were generated carrying smaller, overlapping intervals spanning Iddm26 and fragments of Iddm32 and 33. Analysis of disease susceptibility, age of disease onset, and immune phenotypes in these sublines identified subloci regulating these different parameters. Two ACI.1u.Lyp-derived subloci, Iddm26.1 and Iddm26.2, imparted significant protection from diabetes, decreasing the cumulative incidence by as much as 57% and 28%, respectively. Iddm26.2, which overlaps with the human PTPN22 locus, only affected disease susceptibility, whereas Iddm26.1 also significantly affected disease kinetics, delaying T1D onset by more than 10 days compared with the parental BBDP strain. These Iddm26 subloci also regulated various immune phenotypes, including the proportion of splenic macrophages by Iddm26.1, and the proportion of activated T-cells in secondary lymphoid organs by Iddm26.2. The analysis of Iddm26 congenic animals in two different SPF facilities demonstrated that the influence of this locus on T1D is environment-dependent.

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.

Lactobacillus johnsonii inhibits indoleamine 2,3-dioxygenase and alters tryptophan metabolite levels in BioBreeding rats.

In our previous work, we found that feeding Lactobacillus johnsonii to BioBreeding diabetes-prone (BBDP) rats decreased the incidence of diabetes development. The aim of this study was to investigate host pathways affected by L. johnsonii, with specific focus on the rate-limiting enzyme of tryptophan catabolism, indoleamine 2,3-dioxygenase (IDO). Suspensions of L. johnsonii or an equal volume of vehicle were orally administered to BBDP rats. Tissue IDO was investigated using quantitative RT-PCR and Western blot, whereas tryptophan, kynurenine, and 5-hydroxytryptamine (5-HT) concentrations were quantified by HPLC and ELISA. IDO activity was also investigated using L. johnsonii culture cell-free supernatant (CFS) with affinity-purified IDO and HT-29 intestinal epithelial cells. L. johnsonii feeding resulted in a 17% reduction in serum kynurenine compared with that in vehicle-fed controls, correlating with a 1.4-fold elevation in 5-HT levels. H2O2 produced by L. johnsonii abolished IDO activity in vitro, and L. johnsonii feeding resulted in a 3.9-fold increase in ileum lumen H2O2. L. johnsonii CFS significantly reduced IDO activity in HT-29 intestinal epithelial cells (47% reduction) compared with that in vehicle-treated controls, an effect abolished by catalase treatment. These data support the role of H2O2 in commensal bacteria-host interactions and highlight the influence of commensal bacteria-derived H2O2 on host physiology.

Lowering apolipoprotein CIII delays onset of type 1 diabetes.

Serum levels of apolipoprotein CIII (apoCIII) are increased in type 1 diabetic patients, and when ß cells are exposed to these diabetic sera, apoptosis occurs, an effect abolished by an antibody against apoCIII. We have investigated the BB rat, an animal model that develops a human-like type 1 diabetes, and found that apoCIII was also increased in sera from prediabetic rats. This increase in apoCIII promoted ß-cell death. The endogenous levels of apoCIII were reduced by treating prediabetic animals with an antisense against this apolipoprotein, resulting in a significantly delayed onset of diabetes. ApoCIII thus serves as a diabetogenic factor, and intervention with this apolipoprotein in the prediabetic state can arrest disease progression. These findings suggest apoCIII as a target for the treatment of type 1 diabetes.

Vasopressin induces apical expression of caveolin in rat kidney collecting duct principal cells.

Caveolin (Cav)1 is expressed in the basolateral membrane domain of renal collecting duct (CD) principal cells (PCs), where it is associated with caveolae. To reveal any potential involvement of Cav1 in vasopressin signaling, we used specific monoclonal and polyclonal antibodies to examine its localization in CD PCs of Brattleboro (BB) rats treated with vasopressin (DDAVP). Compared with controls, immunofluorescence revealed a time-dependent increase in Cav1 expression in the apical membrane domain of PCs, where it overlapped with aquaporin-2 (AQP2). After 24 h of DDAVP treatment, Cav1 was visible as an increased number of small apical spots. The staining gradually became more extensive, and, after 2 wk of DDAVP, it occupied the majority of the apical membrane domain of many PCs. Cav1 also assumed an apical localization in PCs of DDAVP-treated Sprague-Dawley and Long-Evans rats. Similarly, Cav2 appeared at the apical pole of PCs after DDAVP treatment of BB, Sprague-Dawley, and Long-Evans rats. Immunogold electron microscopy confirmed bipolar Cav1 membrane expression in DDAVP-treated BB rats, whereas caveolae were only detected on the basolateral membrane. Immunoblot analysis of BB rat whole kidney homogenates revealed no significant increase in Cav1 levels in DDAVP-treated rats, suggesting that DDAVP induces Cav1 relocalization or modifies its targeting. We conclude that Cav1 and Cav2 trafficking and membrane localization are dramatically altered by the action of DDAVP. Importantly, the absence of apical caveolae indicates that while Cavs may have an as yet undetermined role in vasopressin-regulated signaling processes, this is probably unrelated to AQP2 internalization by caveolae.

Inhibition of type 1 diabetes correlated to a Lactobacillus johnsonii N6.2-mediated Th17 bias.

Although it is known that resident gut flora contribute to immune system function and homeostasis, their role in the progression of the autoimmune disease type 1 diabetes (T1D) is poorly understood. Comparison of stool samples isolated from Bio-Breeding rats, a classic model of T1D, shows that distinct bacterial populations reside in spontaneous Bio-Breeding diabetes-prone (BBDP) and Bio-Breeding diabetes-resistant animals. We have previously shown that the oral transfer of Lactobacillus johnsonii strain N6.2 (LjN6.2) from Bio-Breeding diabetes-resistant to BBDP rodents conferred T1D resistance to BBDP rodents, whereas Lactobacillus reuteri strain TD1 did not. In this study, we show that diabetes resistance in LjN6.2-fed BBDP rodents was correlated to a Th17 cell bias within the mesenteric lymph nodes. The Th17 bias was not observed in the non-gut-draining axillary lymph nodes, suggesting that the Th17 bias was because of immune system interactions with LjN6.2 within the mesenteric lymph node. LjN6.2 interactions with the immune system were observed in the spleens of diabetes-resistant, LjN6.2-fed BBDP rats, as they also possessed a Th17 bias in comparison with control or Lactobacillus reuteri strain TD1-fed rats. Using C57BL/6 mouse in vitro assays, we show that LjN6.2 directly mediated enhanced Th17 differentiation of lymphocytes in the presence of TCR stimulation, which required APCs. Finally, we show that footpad vaccination of NOD mice with LjN6.2-pulsed dendritic cells was sufficient to mediate a Th17 bias in vivo. Together, these data suggest an interesting paradigm whereby T1D induction can be circumvented by gut flora-mediated Th17 differentiation.

Genetic dissection of autoimmune type I diabetes in the BB rat.

The BB rat is among the best models of insulin-dependent diabetes mellitus--with onset and pathogenesis closely resembling the human disease. One unusual feature is a severe T-cell lymphopenia, which appears to be inherited as a recessive trait controlled by a single gene, Lyp. Based on genetic analysis of several crosses, we show that development of diabetes involves at least three genes: Lyp, which is tightly linked to the neuropeptide Y (Npy) gene on chromosome 4, a gene linked to the major histocompatibility complex (MHC) on chromosome 20, and a third unmapped gene for which the Fischer rat strain carries an allele conferring resistance.

The immunobiology of apotransferrin in type 1 diabetes.

The transferrin (Tf) family of iron binding proteins includes important endogenous modulators of the immune function that may modulate autoimmune diseases. To define more clearly the role of apotransferrin (apoTf) in type 1 diabetes we determined the impact of this protein on type 1 diabetes as investigated in islet cells, animal models and patient sera. First, we demonstrated that recombinant apoTf counteracts the cytokine-induced death of murine pancreatic islet cells. Secondly, human apoTf administration favourably influences the course of type 1 diabetes in animal models, resulting in protection against disease development that was associated with reduction of insulitis and reduced levels of proinflammatory cytokines. Finally, we confirmed that patients with newly diagnosed type 1 diabetes manifest significantly lower apoTf serum levels compared to healthy controls and patients with long-lasting disease. In conclusion, our data suggest the apoTf pivotal role in the perpetuation of type 1 diabetes pathology.

Potential mechanisms explaining why hydrolyzed casein-based diets outclass single amino acid-based diets in the prevention of autoimmune diabetes in diabetes-prone BB rats.

BACKGROUND: It remains controversial whether avoidance of dietary diabetogenic triggers, such as cow's milk proteins, can prevent type 1 diabetes in genetically susceptible individuals. Here, different extensive casein hydrolysates (HC) and single amino acid (AA) formulations were tested for their effect on mechanisms underlying autoimmune diabetes pathogenesis in diabetes-prone BioBreeding rats. Intestinal integrity, gut microbiota composition and mucosal immune reactivity were studies to assess whether these formulations have differential effects in autoimmune diabetes prevention. METHODS: Diabetes-prone BioBreeding rats received diets in which the protein fraction was exchanged for the different hydrolysates or AA compositions, starting from weaning until the end of the experiment (d150). Diabetes development was monitored, and faecal and ileal samples were collected. Gut microbiota composition and cytokine/tight junction mRNA expression were measured by quantitative polymerase chain reaction. Cytokine levels of ileum explant cultures were measured by ELISA, and intestinal permeability was measured in vivo by lactulose-mannitol assay. RESULTS: Both HC-diet fed groups revealed remarkable reduction of diabetes incidence with the most pronounced effect in Nutramigen®-fed animals. Interestingly, AA-fed rats only showed delayed autoimmune diabetes development. Furthermore, both HC-fed groups had improved intestinal barrier function when compared with control chow or AA-fed animals. Interestingly, higher IL-10 levels were measured in ileum tissue explants from Nutramigen®-fed rats. Beneficial gut microbiota changes (increased Lactobacilli and reduced Bacteroides spp. levels) were found associated especially with HC-diet interventions. CONCLUSIONS: Casein hydrolysates were found superior to AA-mix in autoimmune diabetes prevention. This suggests the presence of specific peptides that beneficially affect mechanisms that may play a critical role in autoimmune diabetes pathogenesis.

Effect of dapoxetine on ejaculatory performance and related brain neuronal activity in rapid ejaculator rats.

INTRODUCTION: A brain network specifically activated when ejaculation occurs has been described in rats. Increasing serotonin (5-hydroxytryptamine [5-HT]) tone impairs ejaculation and chronic 5-HT selective serotonin reuptake inhibitors (SSRIs) are known to inhibit ejaculation. However, efficacy of acute treatment with SSRI varies from one compound to another. The SSRI dapoxetine has been reported to delay ejaculation when given on demand to men with premature ejaculation (PE), although the mechanism of action is unclear. Effects of acute SSRIs on activity of the brain ejaculation circuit in relation with ejaculation have never been examined. AIM: To test the effects of acute administration of the short half-life SSRI dapoxetine on ejaculatory performance and activity in brain ejaculation circuit in rapid ejaculator rats taken as PE model. METHODS: Standard copulatory test was used to attribute one sexual category (sluggish, middle, or rapid) to male rats on the basis of their ejaculatory performance. Parameters of sexual, including ejaculatory, behavior, and Fos level of expression in discrete brain areas were assessed in the three sexual categories and in rapid category following acute oral treatment with dapoxetine. MAIN OUTCOME MEASURES: Ejaculation frequency (EF) and latency (EL) were measured as primary end points of ejaculatory behavior. Density of Fos-immunopositive cells in specific brain areas of brain stem, hypothalamus, and thalamus was determined as marker of neuronal activity. RESULTS: EL and Fos level of expression in hypothalamic and thalamic structures were found related. Dapoxetine acute oral administration (300 mg/kg) to rapid ejaculator rats resulted in (i) diminution of ejaculatory performance (lengthened EL and decreased EF); and (ii) modulation of Fos level of expression in hypothalamic and thalamic nuclei of the brain ejaculatory circuit. CONCLUSION: Acute treatment with dapoxetine, which reduced ejaculatory performance in rapid ejaculator rats, was also accompanied with changes in neuronal activity in components of the brain ejaculatory network.

Type 1 diabetes in BioBreeding rats is critically linked to an imbalance between Th17 and regulatory T cells and an altered TCR repertoire.

Diabetes-prone BioBreeding (DP-BB) rats spontaneously develop type 1 diabetes mellitus (T1DM) on grounds of their MHC haplotype RT1(u) and a point mutation in the Gimap5 gene. In this study, we report that DP-BB rats exhibit an increasingly severe imbalance, in particular between Th17 and regulatory T (T(reg)) cells, within the first months of age. This can be assigned to an excess in effector T cells because neither the percentage nor the function of the T(reg) cells is compromised. Flow cytometric analysis of Vbeta segment usage and CDR3 spectratyping further suggest that the disturbed repertoire of peripheral T cells may also contribute to the development of T1DM in DP-BB rats. Importantly, expansion of T(reg) cells in vivo by means of a CD28 superagonistic Ab as well as adoptive transfer of T(reg) cells efficiently interferes with the development of T1DM in DP-BB rats, whereas treatment with conventional Th cells does not afford protection. Using a newly generated strain of enhanced GFP transgenic rats, we could further demonstrate that the transferred T(reg) cells persist in the recipient rats for several months and partially correct the imbalance between Th17 and T(reg) cells. Thus, our data support the hypothesis that unchecked effector T cell action and a disturbed T cell repertoire contribute to the development of T1DM in DP-BB rats, which may also have implications for a better understanding of the human disease.