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Objective The need of today’s research is to develop successful and reliable diabetic animal models for understanding the disease susceptibility and pathogenesis. Enormous success of animal models had already been acclaimed for identifying key genetic and environmental factors like Idd loci and effects of microorganisms including the gut microbiota. Furthermore, animal models had also helped in identifying many therapeutic targets and strategies for immune-intervention. In spite of a quite success, we have acknowledged that many of the discovered immunotherapies are working on animals and did not have a significant impact on human. Number of animal models were developed in the past to accelerate drug discovery pipeline. However, due to poor initial screening and assessment on inequivalent animal models, the percentage of drug candidates who succeeded during clinical trials was very low. Therefore, it is essential to bridge this gap between pre-clinical research and clinical trial by validating the existing animal models for consistency. Results and Conclusion In this review, we have discussed and evaluated the significance of animal models on behalf of published data on PUBMED. Amongst the most popular diabetic animal models, we have selected six animal models (e.g. BioBreeding rat, “LEW IDDM rat”, “Nonobese Diabetic (NOD) mouse”, “STZ RAT”, “LEPR Mouse” and “Zucker Diabetic Fatty (ZDF) rat” and ranked them as per their published literature on PUBMED. Moreover, the vision and brief imagination for developing an advanced and robust diabetic model of 21st century was discussed with the theme of one mice-one human concept including organs-on-chips.

The depleting Vβ13a T cell receptor monoclonal antibody (mAb) 17D5 prevents both induced and spontaneous autoimmune diabetes in BB rats. Here it was tested in congenic DRLyp/Lyp rats, all of which spontaneously developed diabetes. Starting at 40 days of age, rats were injected once weekly with either saline, His42 Vβ16 mAb, or 17D5 mAb and monitored for hyperglycemia. Diabetes occurred in 100% (n = 5/5) of saline-treated rats (median age, 66 days; range 55–73), and in 100% (n = 6/6) of His42-treated rats (median age, 69 days; range 59–69). Diabetes occurred in fewer (n = 8/11, 73%) 17D5-treated rats at a later age (median 76 days, range 60–92). Three (27%) of the 17D5-treated rats were killed at 101–103 days of age without diabetes (17D5 no-diabetes rats). Survival analysis demonstrated that 17D5 mAb delayed diabetes onset. Saline- and His42-treated rats had severely distorted islets with substantial loss of insulin-positive cells. These rats exhibited prominent hyaluronan (HA) staining, with the intra-islet HA+ accumulations measuring 5,000 ± 2,400 µm2 and occupying 36 ± 12% of islet area, and severe (grade 4) insulitis with abundant infiltration by CD68+, CD3+, and CD8+ cells. The 17D5 mAb-treated rats with delayed diabetes onset exhibited less severe insulitis (predominantly grade 3). In contrast, the 17D5 no-diabetes rats had mostly normal islets, with insulin+ cells representing 76 ± 3% of islet cells. In these rats, the islet HA deposits were significantly smaller than in the diabetic rats; the intra-islet HA+ areas were 1,200 ± 300 µm2 and accounted for 8 ± 1% of islet area. Also, islet-associated CD68+ and CD3+ cells occurred less frequently (on average in 60 and 3% of the islets, respectively) than in the diabetes rats (present in >95% of the islets). No CD8+ cells were detected in islets in all 17D5 no-diabetes rats. We conclude that mAb 17D5 delayed diabetes in DRLyp/Lyp rats and markedly reduced expression of HA and concomitant infiltration of CD68+, CD3+, and CD8+ cells. Our findings underscore the importance of refining immune suppression in prevention or intervention clinical trials to use mAb reagents that are directed against specific T cell receptors.

Retinal homeostasis is under both diurnal and circadian regulation. We sought to investigate the diurnal expression of autophagy proteins in normal rodent retina and to determine if this is impaired in diabetic retinopathy. C57BL/6J mice and Bio-Breeding Zucker (BBZ) rats were maintained under a 12h/12h light/dark cycle and eyes, enucleated over a 24 h period. Eyes were also collected from diabetic mice with two or nine-months duration of type 1 diabetes (T1D) and Bio-Breeding Zucker diabetic rat (BBZDR/wor rats with 4-months duration of type 2 diabetes (T2D). Immunohistochemistry was performed for the autophagy proteins Atg7, Atg9, LC3 and Beclin1. These autophagy proteins (Atgs) were abundantly expressed in neural retina and endothelial cells in both mice and rats. A differential staining pattern was observed across the retinas which demonstrated a distinctive diurnal rhythmicity. All Atgs showed localization to retinal blood vessels with Atg7 being the most highly expressed. Analysis of the immunostaining demonstrated distinctive diurnal rhythmicity, of which Atg9 and LC3 shared a biphasic expression cycle with the highest level at 8:15 am and 8:15 pm. In contrast, Beclin1 revealed a 24-h cycle with the highest level observed at midnight. Atg7 was also on a 24-h cycle with peak expression at 8:15am, coinciding with the first peak expression of Atg9 and LC3. In diabetic animals, there was a dramatic reduction in all four Atgs and the distinctive diurnal rhythmicity of these autophagy proteins was significantly impaired and phase shifted in both T1D and T2D animals. Restoration of diurnal rhythmicity and facilitation of autophagy protein expression may provide new treatment strategies for diabetic retinopathy.

Studies performed in humans and animal models have implicated the environment in the etiology of type 1 diabetes (T1D), but the nature and timing of the interactions triggering β cell autoimmunity are poorly understood. Virus infections have been postulated to be involved in disease mechanisms, but the underlying mechanisms are not known. It is exceedingly difficult to establish a cause-and-effect relationship between viral infection and diabetes in humans. Thus, we have used the BioBreeding Diabetes-Resistant (BBDR) and the LEW1.WR1 rat models of virus-induced disease to elucidate how virus infection leads to T1D. The immunophenotype of these strains is normal, and spontaneous diabetes does not occur in a specific pathogen-free environment. However, β cell inflammation and diabetes with many similarities to the human disease are induced by infection with the parvovirus Kilham rat virus (KRV). KRV-induced diabetes in the BBDR and LEW1.WR1 rat models is limited to young animals and can be induced in both male and female rats. Thus, these animals provide a powerful experimental tool to identify mechanisms underlying virus-induced T1D development.

Type 1 diabetes can have deleterious effects on skeletal muscle and its microvasculature. Our laboratory has recently identified murine double minute-2 as a master regulator of muscle microvasculature by controlling expression levels of two key molecular actors of the angio-adaptive process: the pro-angiogenic vascular endothelial growth factor-A and the anti-angiogenic thrombospondin-1. Here, we show for the first time that in the soleus and plantaris muscles of the diabetes-prone BioBreeding rats, a rodent model of autoimmune type 1 diabetes, murine double minute-2 protein levels are significantly decreased, coinciding with elevated protein levels of thrombospondin-1 and its transcription factor forkhead box O1. Significant capillary regression was observed to similar extent in soleus and plantaris muscles of type 1 diabetic rats. Elevated blood glucose levels were correlated with the loss of capillaries, the reduction in murine double minute-2 expression and with the elevations in thrombospondin-1. Vascular endothelial growth factor-A protein levels were unaltered or even increased in diabetic animals, yet type 1 diabetic animals had less vascular endothelial growth factor receptor-2 abundance. The vascular endothelial growth factor-A/thrombospondin-1 ratio, a good indicator of skeletal muscle angio-adaptive environment, was decreased in type 1 diabetic muscle. Our results suggest that the murine double minute-2-forkhead box O1-thrombospondin-1 pathway plays an important role in angio-regulation of the skeletal muscle in the pathophysiological context of type 1 diabetes.

In spite of the fact that dissolution time profiles of 250mg ursodeoxycholic acid (UCDA) capsules developed by Sponsor and 250mg hard capsules produced by Ursofalk®, Dr. Falk Pharma GmbH, indicated similarity (f2=60.6), a bioavailability study indicated unexpected differences in the formulations. To find an explanation of the in vivo performance of the compared formulations, the dissolution profiles were analyzed using a novel dissolution theory considering: The dissolution model was applied to the measured data using SADAPT. Despite Cmax and AUC values showing higher values after administration of the test product, a reduction of UDCA particle size for the test formulation was suggested for reformulation. The decision was based on the strongly pH-dependent UDCA solubility, formation of insoluble crystals at low pH condition and the known high pH fluctuations ranging from pH1 to 8 in empty stomach. The performed reformulation led to increased dissolution rate of the test product and to a positive bioequivalence study which compared the reformulated test generic formulation with two reference products purchased from two highly regulated markets.

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.

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

Local control of localized protein synthesis Localized protein synthesis provides spatiotemporal precision for injury responses and growth decisions at remote positions in nerve axons. Terenzio et al. show that this process is controlled by local translation of preexisting axonal mRNA encoding the master regulator mTOR (see the Perspective by Riccio). mTOR controls both its own synthesis and that of most newly synthesized proteins at axonal injury sites, thereby determining the subsequent survival and growth of the injured neuron. Science , this issue p. 1416 ; see also p. 1331

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.

Background Genetic and environmental factors both play important roles in the occurrence and progression of diabetic retinopathy (DR). IL-10 592 gene polymorphism is associated with diabetes pathogenesis. This study analyzed the relationship between IL-10 gene promoter-592 loci polymorphism (SNP) in a diabetic model rats with DR. Material/Methods Streptozotocin (STZ) was injected through the tail vein to establish a diabetic rat model. The rats were randomly divided into 2 groups for 3 months’ feeding, including 100 rats in the diabetes-positive control group and 100 rats only injected with citric acid buffer as the blank control group. Fundus fluorescein angiography (FFA) was used to observe retinal vascular changes. Polymerase chain reaction-restriction fragment polymorphisms assay (PCR-RFLP) was used to detect IL-10 gene promoter-592 loci polymorphism in DNA samples. Enzyme-linked immunosorbent assay (ELISA) was performed to test serum IL-10 concentration. Results Serum IL-10 level in DR rats was 33.18±5.0 pg/mL and in the control rats it was 53.33±4.16 pg/mL in (P

Microtubules can regulate GPCR (G protein–coupled receptor) signaling in various cell types. In vascular smooth muscle, activation of the β-adrenoceptor leads to production of cAMP to mediate a vasorelaxation. Little is known about the role of microtubules in smooth muscle, and given the importance of this pathway in vascular smooth muscle cells, we investigated the role of microtubule stability on β-adrenoceptor signaling in rat renal and mesenteric arteries. In isometric tension experiments, incubation with the microtubule inhibitors colchicine and nocodazole enhanced isoprenaline-mediated relaxations of renal and mesenteric arteries that the microtubule stabilizer, paclitaxel, prevented. Sharp microelectrode experiments showed that colchicine treatment caused increased hyperpolarization of mesenteric artery segments in response to isoprenaline. Application of the Kv7 channel blocker, XE991, attenuated the effect of colchicine on isoprenaline relaxations, whereas iberiotoxin—a BKCa channel blocker—had no effect. In addition, colchicine improved the relaxations to the Kv7.2 to 7.5 activator, S-1, in both renal and mesenteric artery segments compared with dimethyl sulfoxide incubation. We determined that increased mesenteric artery myocytes treated with colchicine showed increased Kv7.4 membrane expression, but Western blot analysis showed no change in total Kv7.4 protein. This study is the first to show microtubule disruption improves the β-adrenoceptor–mediated relaxations of mesenteric and renal arteries and determine this enhancement to be because of increased membrane expression of the Kv7 voltage-gated potassium channels.

Type 1 diabetes is associated with abberations of fat metabolism before and after the clinical onset of disease. It has been hypothesized that the absence of the effect of insulin in the liver contributes to reduced hepatic fat synthesis. We measured hepatic gene expression and serum metabolites before and after the onset of hyperglycemia in a BioBreeding rat model of type 1 diabetes. Functional pathway annotation identified that lipid metabolism was differentially expressed in hyperglycemic rats and that these pathways significantly overlapped with genes regulated by insulin. 17 serum metabolites significantly changed in concentration. All but 2 of the identified metabolites had previously been reported in type 1 diabetes, and carbohydrates were overall the most upregulated class of metabolites. We conclude that lack of insulin in the liver contributes to the changes in fat metabolism observed in type 1 diabetes. Further studies are needed to understand the clinical consequences of a lack of insulin in the liver in patients with type 1 diabetes.

BACKGROUND: A complex interplay between a failing intestinal barrier and low-grade inflammation leading to sensorimotor disturbances is an often-cited mechanism in the pathogenesis of functional gastrointestinal disorders (FGID). However, the cause-consequence relationship between these features has not been clearly established. We previously described jejunal alterations in the normoglycemic BB-rat (BBDP-N) model proposing this model as a suitable animal model to study FGID pathophysiology. The current study explores colonic permeability, inflammation, and sensitivity of the BB-rat. METHODS: Colonic tissue of BBDP-N and control (BBDR) rats at 50, 90, 110, 160, and 220 days (n ≥ 7 per group) was used to assess intestinal permeability in Ussing chambers and inflammation, including infiltration by eosinophils, mast cells, and eosinophil peroxidase (EPO) activity. Anxiety-like symptoms were evaluated at 50, 90, and 220 days and colonic sensitivity at 160 and 220 days by measuring the visceromotor response (VMR) to isobaric colorectal distensions. KEYS RESULTS: Lamina propria eosinophil and mast cell infiltration and increased EPO activity were demonstrated from 90 days onward. Increased permeability and myenteric ganglionitis were observed in the oldest BBDP-N rats. At 220 days, the VMR was significantly increased suggesting colonic hypersensitivity. At the same age, increased anxiety-like behavior was observed. CONCLUSION AND INFERENCES: We demonstrated a lamina propria eosinophil and mast cell infiltration preceding visceral hypersensitivity in the colon of the BBDP-N rat, reminiscent of patients with FGID. These findings help elucidating pathogenetic pathways in FGID and further validate the BBDP-N rat as an attractive model to study pathophysiology and therapy of FGID. ispartof: NEUROGASTROENTEROLOGY AND MOTILITY vol:31 issue:7 ispartof: location:England status: published

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.

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.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.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-1β 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.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.

To evaluate the mesenchymal stem cells (MSCs) influence on cytokines and matrix metalloproteinases (MMPs) expression in rat bladder wall regeneration. MSCs cultures from the bone marrow were established. Acellular matrices from the bladder submucosa were prepared. Bladders were reconstructed using cell-seeded (n = 5) and unseeded (n = 5) grafts. MSCs were injected into the bladder wall (n = 5), bladders were incised and MSCs were injected into the circulation (n = 5) or were left intact (n = 5). Animals were killed after 3 months. Bladder histology and immunohistochemical staining of IL-2, IL-4, IL-6, IL-10, TNF-α, TGF-β1, IFN-γ, MMP-2, and MMP-9 were done. Bladders reconstructed with cell-seeded grafts mimicked native tissue, while unseeded grafts revealed shrinkage and morphological irregularities. There were no morphological changes in bladders of other groups. Different pattern of cytokine and MMP expression was observed. Increased expression of anti-inflammatory cytokines and MMPs in bladder promotes detrusor regeneration.

We are exposed to millions of microbial and dietary antigens via the gastrointestinal tract, which likely play a key role in type 1 diabetes (T1D). We differentiated the effects of these two major environmental factors on gut immunity and T1D. Diabetes-prone BioBreeding (BBdp) rats were housed in specific pathogen-free (SPF) or germ-free (GF) conditions and weaned onto diabetes-promoting cereal diets or a protective low-antigen hydrolyzed casein (HC) diet, and T1D incidence was monitored. Fecal microbiota 16S rRNA genes, immune cell distribution, and gene expression in the jejunum were analyzed. T1D was highest in cereal-SPF (65%) and cereal-GF rats (53%) but inhibited and delayed in HC-fed counterparts. Nearly all HC-GF rats remained diabetes-free, whereas HC-fed SPF rats were less protected (7 vs. 29%). Bacterial communities differed in SPF rats fed cereal compared with HC. Cereal-SPF rats displayed increased gut CD3+ and CD8α+ lymphocytes, ratio of Ifng to Il4 mRNA, and Lck expression, indicating T-cell activation. The ratio of CD3+ T cells expressing the Treg marker Foxp3+ was highest in HC-GF and lowest in cereal-SPF rats. Resident CD163+ M2 macrophages were increased in HC-protected rats. The cathelicidin antimicrobial peptide (Camp) gene was upregulated in the jejunum of HC diet–protected rats, and CAMP+ cells colocalized with CD163. A cereal diet was a stronger promoter of T1D than gut microbes in association with impaired gut immune homeostasis.

Background/aims: Type 1 diabetes is often accompanied by gastrointestinal motility disturbances. Vagal neuropathy, hyperglycemia, and alterations in the myenteric plexus have been proposed as underlying mechanism. We therefore studied the relationship between vagal function, gastrointestinal motiliy and characteristics of the enteric nervous system in the biobreeding (BB) rat known as model for spontaneous type 1 diabetes. Methods: Gastric emptying breath test, small intestinal electromyography, relative risk-interval variability, histology and immunohistochemistry on antral and jejunal segments were performed at 1, 8 and 16 weeks after diabetes onset and on age-matched controls. Results: We observed no consistent changes in relative risk-interval variability and gastric emptying rate. There was however, a loss of phases 3 with longer duration of diabetes on small intestinal electromyography. We found a progressive decrease of nitrergic neurons in the myenteric plexus of antrum and jejunum, while numbers of cholinergic nerve were not altered. In addition, a transient inflammatory infiltrate in jejunal wall was found in spontaneous diabetic BB rats at 8 weeks of diabetes. Conclusions: In diabetic BB rats, altered small intestinal motor control associated with a loss of myenteric nitric oxide synthase expression occurs, which does not depend on hyperglycemia or vagal dysfunction, and which is preceded by transient intestinal inflammation.

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.

PURPOSE To evaluate the capabilities of gelatin derivatives that incorporate phenolic hydroxyl groups (gelatin-Ph) as a delivery carrier with recombinant human platelet-derived growth factor-BB (rhPDGF-BB) for bone regeneration. MATERIALS AND METHODS The growth factor release profile from gelatin-Ph gel or atelocollagen was analyzed to evaluate the capability of gelatin-Ph as a carrier. The biocompatibility of gelatin-Ph (ie, the survival rates of the cells during gelation, and the morphology and proliferation of the cells) were investigated with rat bone marrow cells (RBMCs) in vitro. To evaluate the effect of the gelatin-Ph-rhPDGF-BB complex on bone formation, the complex was applied to bone defects in rat tibiae and undecalcified specimens were fabricated for histologic analysis. RESULTS Scarce amounts of rhPDGF-BB from gelatin-Ph gel were detected during 30 days, but the addition of protease induced the release of rhPDGF-BB on day 31. No differences were observed in the survival rates of RBMCs during gelation and in the morphology and proliferation of RBMCs on the gel sheet between groups. Histological analyses demonstrated that the complex enhanced bone formation and mineralization at 2 and 4 weeks. CONCLUSION These results suggest that gelatin-Ph gel can be an ideal delivery carrier, and the localized delivery of rhPDGF-BB in gelatin-Ph can contribute to bone formation.

Background Poor bone quality, increased fracture risks, and impaired bone healing are orthopedic comorbidities of type 1 diabetes (T1DM). Standard osteogenic growth factor treatments are inadequate in fully rescuing retarded healing of traumatic T1DM long bone injuries where both periosteal and bone marrow niches are disrupted. We test the hypotheses that osteogenesis of bone marrow-derived stromal cells (BMSCs) and periosteum-derived cells (PDCs), two critical skeletal progenitors in long bone healing, are both impaired in T1DM and that they respond differentially to osteogenic bone morphogenetic proteins (BMPs) and/or insulin-like growth factor-1 (IGF-1) rescue. Methods BMSCs and PDCs were isolated from Biobreeding Diabetes Prone/Worcester rats acquiring T1DM and normal Wistar rats. Proliferation, osteogenesis, and adipogenesis of the diabetic progenitors were compared with normal controls. Responses of diabetic progenitors to osteogenesis rescue by rhBMP-2/7 heterodimer (45 or 300 ng/ml) and/or rhIGF-1 (15 or 100 ng/ml) in normal and high glucose cultures were examined by alizarin red staining and qPCR. Results Diabetic BMSCs and PDCs proliferated slower and underwent poorer osteogenesis than nondiabetic controls, and these impairments were exacerbated in high glucose cultures. Osteogenesis of diabetic PDCs was rescued by rhBMP-2/7 or rhBMP-2/7 + rhIGF-1 in both normal and high glucose cultures in a dose-dependent manner. Diabetic BMSCs, however, only responded to 300 ng/nl rhBMP-2/7 with/without 100 ng/ml rhIGF-1 in normal but not high glucose osteogenic culture. IGF-1 alone was insufficient in rescuing the osteogenesis of either diabetic progenitor. Supplementing rhBMP-2/7 in high glucose osteogenic culture significantly enhanced gene expressions of type 1 collagen (Col 1), osteocalcin (OCN), and glucose transporter 1 (GLUT1) while suppressing that of adipogenic marker peroxisome proliferator-activated receptor gamma (PPARγ) in diabetic PDCs. The same treatment in high glucose culture only resulted in a moderate increase in Col 1, but no significant changes in OCN or GLUT1 expressions in diabetic BMSCs. Conclusions This study demonstrates more effective osteogenesis rescue of diabetic PDCs than BMSCs by rhBMP-2/7 with/without rhIGF-1 in a hyperglycemia environment, underscoring the necessity to tailor biochemical therapeutics to specific skeletal progenitor niches. Our data also suggest potential benefits of combining growth factor treatment with blood glucose management to optimize orthopedic therapeutic outcomes for T1DM patients.

Spontaneous type 1 diabetes in BB rats is dependent on the RT1u MHC haplotype and homozygosity for an allele at the Lyp locus, which is responsible for a peripheral T-lymphopenia. Genetic studies have shown that there are other, as yet unidentified, genetic loci contributing to diabetes susceptibility in this strain. BB rats carrying wild-type Lyp alleles are not lymphopenic and are resistant to spontaneous diabetes (DR). Here we show that thymectomy and exposure to one sublethal dose of γ-irradiation (TX-R) at 4 weeks of age result in the rapid development of insulitis followed by diabetes in 100% of DR rats. Administration of CD4+45RC− T-cells from unmanipulated, syngeneic donors immediately after irradiation prevents the disease. Splenic T-cells from TX-R-induced diabetic animals adoptively transfer type 1 diabetes to T-deficient recipients. ACI, WF, WAG, BN, LEW, PVG, and PVG.RT1u strains are resistant to TX-R-induced insulitis/diabetes. Genetic analyses revealed linkage between regions on chromosomes 1, 3, 4, 6, 9, and 16, and TX-R-induced type 1 diabetes in a cohort of nonlymphopenic F2 (Wistar Furth × BBDP) animals. This novel model of TX-R-induced diabetes in nonlymphopenic BB rats can be used to identify environmental and cellular factors that are responsible for the initiation of antipancreatic autoimmunity.

Nitric oxide (NO) synthesis in endothelial cells is impaired in diabetes. We previously showed that impaired NO synthesis in the spontaneously diabetic BB (BBd) rat is due to decreased levels of tetrahydrobiopterin (BH4), secondary to decreased expression of GTP cyclohydrolase I (GTPCH). The aim of this study was to utilize adenoviral GTPCH gene transfer to reverse BH4 deficiency and repair the ability of endothelial cells to produce NO. GTPCH gene transfer increased BH4 levels in BBd endothelial cells from 0.17 +/- 0.02 (mean +/-SE) to 73.37 +/- 14.42 pmol/million cells and NO production from 0.77 +/- 0.07 to 18.74 +/- 5.52 nmol/24 h/million cells. To demonstrate a functional effect of increasing BH4 concentrations in tissues, we transferred GTPCH into aortic rings from BBd and Zucker diabetic fatty (ZDF) rats, models of human type I and type II diabetes, respectively. GTPCH gene transfer led to a dose-dependent increase in acetylcholine-induced vasorelaxation, preventable by inhibiting NO synthase. Maximal relaxation of virus-treated rings (10(10) virus particles/ml) to acetylcholine was significantly higher than sham-treated rings (BBd 64% vs. 37%, P

We recorded arterial pressure (BP) and heart rate (HR) in type-1 diabetic rats vs. controls for > 6 months. Diabetic rats (DIAB) were maintained on insulin from the day glucose >250 mg/dl (“Day 0”). Weight was similar between groups until ~ 3 weeks before Day 0 when the weight in DIAB transiently lagged the controls (CONT); this difference was maintained throughout the study, but both groups otherwise gained weight in parallel. Plasma glucose attained 371 ± 109 (SD) mg/dl by day 1 in DIAB. Mean BP was similar across groups, and declined through the initial 4–6 months in both the CONT (at − 0.06 ± 0.04 mm Hg/day) and in the DIAB (at − 0.14 ± 0.21 mm Hg/day; NS vs. CONT). HR in the CONT (Month 1: 341 ± 13 bpm) exceeded DIAB (325 ± 25 bpm) through ~ 6 months after Day 0, and also decreased progressively over this period in CONT (− 0.19 ± 0.14 bpm/day) and DIAB (− 0.29 ± 0.23 bpm/day; NS vs. CONT) before leveling. The BP power within 0.35–0.45 Hz changed during the 90 min before vs. after the transition from dark to light, and light to dark; there were no between group differences. The slope of the log–log linear portion of the BP power spectrum between 1.0/h and 1/min was similar across groups, and increased in both from month 1 to month 6. Regulatory mechanisms maintain similar profiles in BP and HR in diabetic vs. control animals through the initial half year of the disease.

The aim of this study was to evaluate nerve regeneration in relation to the transcription factor, Activating Transcription Factor 3 (ATF 3), and an apoptotic marker, caspase 3, in the Schwann cells of diabetic BB rats (i.e. display type 1 diabetes phenotype). Sciatic nerves in healthy Wistar rats and in diabetic BB rats were transected and immediately repaired. Axonal outgrowth (neurofilament staining) and expression of ATF 3 and caspase 3 were quantified by immunohistochemistry after six days. There was no difference in axonal outgrowth between healthy and diabetic rats. However, the sciatic nerve in the diabetic rats exhibited a larger number of ATF 3 expressing Schwann cells at the site of the lesion and also a higher number of caspase 3 expressing Schwann cells. Similar differences were observed in the distal nerve segment between the healthy and diabetic rats. There were no correlations between the number of Schwann cells expressing ATF 3 and caspase 3. Thus, diabetic BB rats display an increased activation of ATF 3 and also a rise in apoptotic caspase 3 expressing Schwann cells, but with no discrepancy in length of axonal outgrowth after nerve injury and repair at six days. Knowledge about signal transduction mechanisms in diabetes after stress may provide new insights into the development of diabetic neuropathy and neuropathic pain.

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.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.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 KIn 2 rate models of diabetes, we found that activation of the TRESK K

Scope: To identify genes involved in the susceptibility to iodine-induced autoimmune thyroiditis. Methods and results: Diabetes, thyroiditis-prone (BBdp) and -resistant (BBc) rats were fed either a control or a high-iodine diet for 9 wk. Excess iodine intake increased the incidence of insulitis and thyroiditis in BBdp rats. BBdp rats fed the high-iodine diet that did not develop thyroiditis had higher mRNA levels of Fabp4, Cidec, perilipin, Pparγ and Slc36a2 than BBdp rats fed the control diet and BBc rats fed either the control or the high-iodine diet. BBdp rats fed the high-iodine diet that did develop thyroiditis had higher mRNA levels of Cidec, Icam1, Ifitm1, and Slpi than BBdp rats fed the control diet and BBc rats fed either the control or the high-iodine diet. BBdp rats that did develop thyroiditis had lower mRNA levels of Fabp4, perilipin and Slc36a2 but higher mRNA levels of Icam1, Ifitm1 and Slpi than BBdp that did not develop thyroiditis. Excess dietary iodine also increased the protein levels of Fabp4, Cidec and perilipin in BBdp rats. Conclusion: Differential expression of thyroid genes in BBdp versus BBc rats caused by excess dietary iodine may be implicated in autoimmune thyroiditis and insulitis pathogenesis.

Background Rat models of diabetes have emerged as a powerful experimental tool for addressing the role of microbial pathogens in the mechanism of autoimmune diabetes. We have used the biobreeding diabetes resistant and LEW1.WR1 rat models to identify the role of virus-induced innate immunity in the mechanism of type 1 diabetes. Methods Groups of rats 21–25 days of age were left untreated, injected i.p. with 1 × 107 PFU of Kilham rat virus (KRV) only, or with 1–3 µg/g body-weight-purified toll-like receptor agonists on three consecutive days and infected with 1 × 107 PFU of KRV on the following day. Spleens and pancreatic lymph nodes were recovered 5 days after infection and used for gene array analysis. To test the role of inflammation in diabetes, rats injected with KRV only or Poly(I:C) plus KRV were also administered with 2 or 0.2 µg/g body weight of dexamethasone and followed for diabetes for 40 days. Results KRV induced the expression of a vast array of proinflammatory genes in pancreatic lymph nodes on day 5 following infection. Brief dexamethasone therapy downmodulated inflammation and completely blocked diabetes. Conclusions Our data suggest a strong association between early virus-induced proinflammatory responses and islet destruction and raise the possibility that targeting innate immune pathways in the early stages of diabetes may be a useful strategy for disease prevention. Copyright © 2011 John Wiley & Sons, Ltd.

Aims: The Gimap gene family has been shown to be integral to T cell survival and development. A frameshift mutation in Gimap5, one of seven members of the Gimap family, results in lymphopenia and is a prerequisite for spontaneous type 1 diabetes (T1D) in the BioBreeding (BB) rat. While not contributing to lymphopenia, the Gimap family members proximal to Gimap5, encompassed within the Iddm39 quantitative trait locus (QTL), have been implicated in T1D. We hypothesized that expression of the Gimap family members within the Iddm39 QTL, during thymocyte development as well as in peripheral T and B cells contribute to T1D. Main methods: Cell sorted subpopulations were analyzed by quantitative real time (qRT) PCR. Key findings: Gimap4 expression was reduced in DR.(lyp/lyp) rat double negative, double positive and CD8 single positive (SP) thymocytes while expression of Gimap8, Gimap6, and Gimap7 was reduced only in CD8 SP thymocytes. Interestingly, expression of the entire Gimap gene family was reduced in DR.(lyp/lyp) rat peripheral T cells compared to non-lymphopenic, non-diabetic DR.(+/+) rats. With the exception of Gimap6. the Gimap family genes were not expressed in B cells from spleen and mesenteric lymph node (MLN). Expression of Gimap9 was only detected in hematopoietic cells of non B cell lineage such as macrophage, dendritic or NK cells. Significance: These results suggest that lack of the Gimap5 protein in the DR.(lyp/lyp) congenic rat was associated with impaired expression of the entire family of Gimap genes and may regulate T cell homeostasis in the peripheral lymphoid organs. (C) 2011 Elsevier Inc. All rights reserved.

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.

BB rats develop type 1 diabetes and WOKW rats facets of the metabolic syndrome. Both strains are common the RT1 (u) haplotype of major histocompatibility complex (MHC) which is essential for type 1 diabetes development in BB rats ( IDDM1). However, BB rats need an additional gene (lymphopenia, IDDM2, GIMAP5) to develop type 1 diabetes. Because WOKW lacks IDDM2 and does not develop hyperglycemia a congenic WOKW rat strain was generated recombining the region of chromosome 4 with IDDM2 onto the genetic background of WOKW rats (WOKW.4BB). These newly established rats and their parental WOKW rats were genetically and phenotypically characterized. Congenic WOKW.4BB rats showed a lymphopenic phenotype. The sequences of the highly polymorphic exon 2 of RT1-BB class II gene in WOKW, BB/OK, WOKW.4BB and LEW.1W rats were comparable and clearly showed the RT1 (u) haplotype. In addition, there were significant differences in metabolic traits between WOKW.4BB and parental WOKW. Although congenic WOKW.4BB rats were homozygous for IDDM1 and IDDM2 of the BB/OK rat none of WOKW.4BB rats developed hyperglycemia. This observation may be attributed to the idea that either WOKW.4BB rats need a third IDDM gene of BB/OK rats to develop hyperglycemia or WOKW background gene/s protect/s them for hyperglycemia.

Serum levels of apolipoprotein CIII (apoCIII) are increased in type 1 diabetic patients, and when β 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 β-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.

Objectives An intestinal permeability defect precedes type 1 diabetes mellitus and may be a permissive factor in its pathogenesis. Butyrate strengthens the intestinal tight junctions. We hypothesized that enteral administration of sodium butyrate (NaB) in preweaned rats would result in differences in the development of diabetes associated with decreased inflammation and pancreatic β-cell destruction. Materials and methods Using biobreeding diabetes-prone rat pups, oral NaB or saline was administered twice per day via micropipette from postnatal days 10 to 23. Rat pups were randomly assigned to 1 of 4 groups for the first experiment (control group, n = 7) and 3 different doses of butyrate groups (n = 8 for each group) and 2 groups for the second and third experiments (control n = 23; NaB at 400 mg · kg(-1) · day(-1), n = 20). Animals were studied into adulthood (up to day 140) for development of diabetes. Results The results showed that the survival rates were 28% versus 20% (butyrate vs control). No significant differences in survival were seen; however, there was a trend of delaying of onset of diabetes in the butyrate group. There were no differences of pancreatic histology score of islet inflammation between the 2 groups. Cytokine-induced neutrophil chemoattractant-1 was lower in the butyrate group at a dose of 400 mg · kg(-1) · day(-1) in the distal small intestine (P = 0.008) and in the liver (P = 0.01). There were no significant differences in the tracer flux measurements across the distal ileum and colon between the 2 animal groups. Conclusions Oral NaB given during the preweaning period did not significantly decrease the subsequent development of death from diabetes in biobreeding diabetes-prone rats.

We have recently found that 3T3-L1 adipocytes secrete microvesicles, known as adipocyte-derived microvesicles (ADMs), with angiogenic activity. In this study, we found that ADMs contain RNA without typical 28S and 18S ribosomal RNA inside the vesicles. Microarray analysis revealed that ADMs contain approximately 7000 mRNAs and 140 microRNAs. Most of transcripts for adipocyte-specific and dominant genes were contained in the ADMs, and their abundance was mostly correlated with that in the donor cells. Abundance of adipocyte-related microRNAs was also mostly correlated with that in the donor cells. ADMs mediated transport of adiponectin and resistin gene transcripts into RAW264.7 macrophages. Moreover, adipocyte-specific gene transcripts such as adiponectin, resistin, and PPARgamma2 were found in microvesicles isolated from rat serum. Thus, ADM might play a role as a novel intercellular communication tool by transporting RNA in paracrine and possibly endocrine manners.

The clinical development of a candidate p38 kinase inhibitor was terminated because of its unexpectedly rapid clearance in human subjects. Its short half-life and metabolic profile in human beings were vastly different from that in rats, dogs, and monkeys characterized during routine pre-clinical studies. Mice generated the predominant drug (4-hydroxylated) metabolite produced in human beings, which was not found in other species. The data from a murine in vitro drug biotransformation assay that used liver extracts from 14 inbred mouse strains were analyzed by haplotype-based computational genetic analysis. This led to the identification of aldehyde oxidase-1 (AOX1) as the enzyme responsible for the rapid metabolism of this drug. Specific enzyme inhibitors and expressed recombinant enzymes were used to confirm that AOX catalyzed the formation of the 4-hydroxylated drug metabolite in mouse and man. Genetic variation within Aox1 regulated the level of hepatic Aox1 mRNA, AOX1 protein, and enzyme activity among the inbred strains. Thus, computational murine pharmacogenetic analysis can facilitate the identification and characterization of drug metabolism pathways that are differentially utilized by humans and other species.

Type 1 diabetes (T1D) mellitus can best be characterized as a disorder of gluco-regulation due to the insufficient production of a single critical hormone: insulin. Since the middle of the last century the most efficient pharmacologic solution has been to administer the hormone to the patient daily. Increasingly sophisticated dosing schedules together with the availability of recombinant variants of the hormone have succeeded in granting normal lifespan to type 1 diabetics. Nevertheless, no matter the degree of sophistication, current even aggressive regimens have not proven capable of faithfully recapitulating the normal performance of the endogenous insulin producing beta cells in response to glucose. This limit leads to the inevitable principal causes of morbidity and mortality associated with T1D, namely the complications of kidney and heart together with ocular and neural diseases. While insulin replacement continues to be the primary treatment, the need to establish physiologic gluco-regulation in order to avoid complications has led to multiple avenues of alternative interventions, most of which are at the experimental stage. What all of these interventions have in common, however, is the hurdle imposed by the immune system at the level of ongoing autoimmunity and, in some cases, at the level of transplant rejection by the host. Autoimmunity in T1D is characterized by an inflammatory response against the insulin-producing beta cells of the pancreas, a chronic inflammation around and in the pancreatic islets of Langerhans termed “peri-insulitis” and “insulitis”, respectively. As studied in the two classic rodent models of the disease (diabetes-prone BioBreeding, DP-BB, rat and non obese diabetic, NOD, prone mouse), early on in the acute phase of the immune attack the islets exhibit an abundant cell infiltration by mononuclear cells, macrophages and dendritic cells (DC). With time, T-cells become the major constituent of the insulitis and are responsible for the greatest beta cell damage and destruction. The clinical onset manifests once the number of surviving beta cells cannot secrete sufficient insulin to satisfy the body’s needs. A strong genetic predisposition is a conditio sine qua non of T1D and a large body of studies support that key genetic susceptibility loci affect the genesis, function and survival of immune cell subsets. To understand the critical role played by the genetic predisposition in T1D, it is necessary to consider the processes that shape Diabetes mellitus hoy

This study investigates the potential of 3D silk scaffolds fabricated using tropical tasar non-mulberry, Antheraea mylitta and mulberry, Bombyx mori silk gland fibroin proteins as substrate for osteogenic and adipogenic differentiation of rat bone marrow cells (BMCs). The scaffolds are mechanically robust and show homogenous pore distribution with high porosity and interconnected pore walls. Low immunogenicity of fabricated silk scaffolds as estimated through TNF α release indicates its potential as future biopolymeric graft material. Rat bone marrow cells cultured on scaffolds for 28 days under static conditions in osteogenic and adipogenic media respectively led to induction of differentiation. Proliferation and spreading of fibroblasts and bone marrow cells on silk scaffolds were observed to be dependent on scaffold porosity as revealed through confocal microscopic observations. Histological analysis shows osteogenic differentiation within silk scaffolds resulting in extensive mineralization in the form of deposited nodules as observed through intense Alizarin Red S staining. Similarly, adipogenesis was marked by the presence of lipid droplets within scaffolds on staining with Oil Red O. Real-time PCR studies reveal higher transcript levels for osteopontin (Spp1), osteocalcin (Bglap2) and osteonectin (Sparc) genes under osteogenic conditions. Similarly, upregulated adipogenic gene expression was observed within A. mylitta and B. mori scaffolds under adipogenic conditions for Peroxisome proliferator activated receptor gamma (PPARγ2), lipoprotein lipase (LPL) and adipocyte binding protein (aP2) genes. The results suggest suitability of silk fibroin protein 3D scaffolds as natural biopolymer for potential bone and adipose tissue engineering applications.

Thioglycosides and C-glycosides have received considerable attention, because they are widely employed as biological inhibitors, inducers and ligands for affinity chromatography of carbohydrate-processing enzymes and proteins. Moreover, they are promising candidates in synthetic carbohydrate chemistry as convenient and versatile glycosyl donors. Among these glycosyl donors are the thioglycosyl and N-glycosyl heterocycles that are sufficiently stable under a variety of reaction conditions and have the ability to be readily converted into a variety of other functionalities. We report here, the synthesis of 2-thioxo-quinazolines 1a-c which were used as a base to the synthesis of S-nucleoside of types 10, 11 and acyclic C-nucleoside analogs of type 14 and their analgesic and anti-inflammatory activities were evaluated giving good results.

Diabetes mellitus is a common systemic disease that has been associated with poor fracture healing outcomes. The mechanism through which diabetes impairs bone regeneration is unknown. One possible mechanism may be related to either decreased or uncoordinated release of local growth factors at the fracture site. Indeed, previous studies have found reduced platelet-derived growth factor (PDGF) levels in the fracture callus of diabetic rats, suggesting that local application of PDGF may overcome the negative effects of diabetes and promote fracture healing. To test this hypothesis, low (22 microg) and high (75 ug) doses of recombinant human PDGF-BB (rhPDGF-BB) were applied directly to femur fracture sites in BB Wistar diabetic rats that were then compared to untreated or vehicle-treated animals. rhPDGF-BB treatment significantly increased early callus cell proliferation compared to that in control specimens. Low dose rhPDGF-BB treatment significantly increased callus peak torque values (p < 0.05) at 8 weeks after fracture as compared to controls. High dose rhPDGF-BB treatment increased callus bone area at 12 weeks postfracture. These data indicate that rhPDGF-BB treatment ameliorates the effects of diabetes on fracture healing by promoting early cellular proliferation that ultimately leads to more bone formation. Local application of rhPDGF-BB may be a new therapeutic approach to treat diabetes-impaired fracture healing.

Type 1 diabetes (T1D) occurs when the immune system attacks the insulin-producing beta cells located in the islets of Langerhans within the pancreas. Animal models have played a prominent role in developing an understanding of this disease process, through studies of genetic susceptibility, progression of hyperglycemia, and novel approaches to therapy. Here, we critically evaluate the currently available diabetic animal models and their propensity to match and predict disease outcomes in man as well as propose new in vitro and in vivo systems that may facilitate progress in the T1D field.

The fact that insulitis occurs also in normoglycaemic BB rats led us to investigate the phenotypes of lymphocytes invading the pancreatic islets of prediabetic BB/OK rats in comparison to age- and sex-matched normoglycaemic animals in a retrospective analysis. By using a panel of monoclonal antibodies we investigated the number of pan T-cells, T-helper cells, cytotoxic T-cells and NK-cells and determined the number of activated cells by measurement of class I, class II and IL-2 receptor positive cells. The bound primary antibodies were visualized using the APAAP-technique. The prediabetic rats showed a significantly decreased pancreatic insulin content which was drastically reduced at diagnosis of diabetes. This was accompanied by reduction of the B-cell volume density. The prediabetic as well as the long-term normoglycaemic BB rats showed an accumulation of mononuclear cells (all phenotypes investigated) within the pancreatic islets. Concerning the phenotypes of infiltrating cells there was no qualitative difference between long-term normoglycaemic and prediabetic rats but quantitatively an enhanced amount of W3/25+, OX-8+, OX-6+ and ART-18+ cells could be observed in the prediabetic animals. From our results we conclude that an immunological B-cell destructive process occurs also in long-term normoglycaemic BB rats by participation of mononuclear cells qualitatively not different from those observed in prediabetic animals. Activated T-cells (OX-19+, OX-8+, W3/25+) expressing class II antigens (OX-6+) and the IL-2 receptor (ART-18+) seem to play a significant role in the amplified immunological pancreatic B-cell destruction.

The intact pancreatic islet can be destroyed by antibody-dependent cell-mediated cytotoxicity (ADCC). T cell-mediated cytotoxicity (CMC) might additionally be important in the pathogenesis of IDDM. However, in vitro alterations of islets due to CMC have so far not been demonstrated. For the evaluation of the cytotoxic attack caused by ADCC and CMC against the islets in the development of IDDM we used a BB rat line with a high incidence of the disease (dp BB/OK). Cytotoxicity tests were carried out on autologous and on nonsyngeneic islets with mononuclear spleen cells and serum from non-diabetic BB rats of different ages. The cytotoxic action of the mononuclear cells was quantified by the specific 51Cr-release from the islets after pretreatment with serum. It was found that an anti-islet cytotoxicity appears with a peak incidence between 40 and 60 days of age. The frequency of cytotoxicity in vitro was related to the incidence of diabetes as normally observed in this rat line. Furthermore, it was shown that both autologous, allogeneic and xenogeneic islets can be destroyed by mononuclear spleen cells and serum of dp BB/OK rats.--The frequency and the strength of anti-islet cytotoxicity in vitro were higher in these dp BB/OK animals than in a control group of non-diabetes prone BB/PhiK rats. There was an association between cytotoxic 51Cr-release in the positive assays and a reduction in the hormone content of pancreatic islets.--This report provides evidence of cell-mediated immune damage of islets during the prediabetic state of BB rats suggesting that both CMC and ADCC are involved in islet cell killing.