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A central focus of research in the type 1 diabetes (T1D) field is to develop ways to safely improve [3-cell survival and function and promote their replication. The addition of [...], γ-Aminobutyric acid (GABA) has been shown to inhibit apoptosis of rodent β-cells in vitro. In this study, we show that activation of [GABA.sub.A] receptors ([GABA.sub.A]-RS) or [GABA.sub.B]-RS significantly inhibits oxidative stress-related β-cell apoptosis and preserves pancreatic β-cells in streptozotocin-rendered hyperglycemic mice. Moreover, treatment with GABA, or a [GABA.sub.A]-R- or [GABA.sub.B]-R-specific agonist, inhibited human β-cell apoptosis following islet transplantation into NOD/scid mice. Accordingly, activation of [GABA.sub.A]-RS and/or [GABA.sub.B]-Rs may be a useful adjunct therapy for human islet transplantation. GABA-R agonists also promoted β-cell replication in hyperglycemic mice. While a number of agents can promote rodent β-cell replication, most fail to provide similar activities with human β-cells. In this study, we show that GABA administration promotes β-cell replication and functional recovery in human islets following implantation into NOD/scid mice. Human β-cell replication was induced by both [GABA.sub.A]-R and [GABA.sub.B]-R activation. Hence, GABA regulates both the survival and replication of human [3-cells. These actions, together with the anti-inflammatory properties of GABA, suggest that modulation of peripheral GABA-Rs may represent a promising new therapeutic strategy for improving [3-cell survival following human islet transplantation and increasing [3-cells in patients with diabetes. Diabetes 62:3760-3765, 2013

The expression of imaging reporter genes in β-cells has provided powerful tools for studying β-cell development, embryonic and adult stem cell differentiation into β-cells, transdifferentiation, and islet transplantation (1-13). Bioluminescent [...], OBJECTIVE--β-Cells that express an imaging reporter have provided powerful tools for studying β-cells development, islet transplantation, and β-Cells autoimmunity. To further expedite diabetes research, we generated transgenic C57BL/6 'MIP-TF' mice that have a mouse insulin promoter (MIP) driving the expression of a trifusion (TF) protein of three imaging reporters (luciferase/ enhanced green fluorescent protein/HSVl-sr39 thymidine kinase) in their β-cells. This should enable the noninvasive imaging of β-cells by charge-coupled device (CCD) and micro-positron emission tomography (PET), as well as the identification of β-cells at the cellular level by fluorescent microscopy. RESEARCH DESIGN AND METHODS--MIP-TF mouse β-Cells were multimodality imaged in models of type 1 and type 2 diabetes. RESULTS--MIP-TF mouse β-cells were readily identified in pancreatic tissue sections using fluorescent microscopy. We show that MIP-TF β-cells can be noninvasively imaged using microPET. There was a correlation between CCD and microPET signals from the pancreas region of individual mice. After low-dose streptozotocin administration to induce type 1 diabetes, we observed aprogressive reduction in bioluminescence from the pancreas region before the appearance of hyperglycemia. Although there have been reports of hyperglycemia inducing proinsulin expression in extrapancreatic tissues, we did not observe bioluminescent signals from extrapancreatic tissues of diabetic MIP-TF mice. Because MIP-TF mouse β-cells express a viral thymidine kinase, ganciclovir treatment induced hyperglycemia, providing a new experimental model of type 1 diabetes. Mice fed a high-fat diet to model early type 2 diabetes displayed a progressive increase in their pancreatic bioluminescent signals, which were positively correlated with area under the curve-intraperitoneal glucose tolerance test (AUC-IPGTT). CONCLUSIONS--MIP-TF mice provide a new tool for monitoring β-cells from the single cell level to noninvasive assessments of β-cells in models of type 1 diabetes and type 2 diabetes. Diabetes 60:1383-1392, 2011

OBJECTIVE--To study how tolerance to GAD65 affects the development of autoimmunity to other β-cell autoantigens (β-CAAs) in GAD654ransgenic (GAD-tg) NOD mice. RESEARCH DESIGN AND METHODS--We used ELISPOT to characterize the frequency and functional phenotype of T-cell responses to GAD65 and other β-CAAs at different ages in GAD-tg mice and their NOD mouse littermates. RESULTS--In young GAD-tg mice, Th1 responses to GAD65's dominant determinants were 13-18% of those in young NOD mice. This coincided with a great reduction in Th1 responses to other β-CAAs. Evidently, GAD65-reactive T-cells are important for activating and/or expanding early autoreactivities in NOD mice. As GAD-tg mice aged, their T-cell responses to GAD65 remained low, but they developed supernormal splenic and pancreatic lymph node T-cell autoimmunity to other β-CAAs. Apparently, the elimination/impairment of many GAD65-reactive T-cells allowed other β-CAA-reactive T-cells to eventually expand to a greater extent, perhaps by reducing competition for antigen-presenting cells, or homeostatic proliferation in the target tissue, which may explain the GAD-tg mouse's usual disease incidence. CONCLUSIONS--Transgenically induced reduction of GAD65 autoreactivity curtailed the development of early T-cell responses to other β-CAAs. However, later in life, β-CAA-reactive T-cells expanded to supernormal levels. These data suggest that early p-cell autoreactivities are mutually dependent for support to activate and expand, while later in the disease process, autoantigen-specific T-cell pools can expand autonomously. These findings have implications for understanding type 1 diabetes immunopathogenesis and for designing antigen-based immunotherapeutics., The role of T-cell autoreactivity to the 65-kDa form of GAD (GAD65) in the etiology of type 1 diabetes in nonobese diabetic (NOD) mice has been much debated, in large [...]

Antigen-based therapies (ABTs) seek to prevent or inhibit autoimmune diseases by inducing regulatory T-cell responses (active tolerance) or anergizing/deleting pathogenic T-cells (passive tolerance). The theoretical appeal of this therapeutic approach [...]

Reactive oxygen species, such as superoxide, and nitrogen oxides, such as peroxynitrite, are thought to contribute to β-cell destruction during the disease process that leads to type 1 diabetes. EUK-8 is a member of a new class of synthetic salen-manganese compounds with low toxicity that possess catalytic superoxide dismutase, peroxidase, and catalase activity that can inactivate superoxide and nitrogen oxides (e.g., peroxynitrite and nitrogen dioxide). We observed that EUK-8 administration inhibited the adoptive transfer of type 1 diabetes to NOD mice. In addition, administration of EUK-8 to NOD mice with established autoimmunity completely prevented the development of type 1 diabetes for up to 1 year in age, even though the treatment was discontinued after 35 weeks of age. EUK-8 treatment also prolonged the survival of islet allografts in newly diabetic NOD mice. Thus, reactive oxygen and nitrogen species contribute to the pathoetiology of both spontaneous type 1 diabetes and allograft rejection. In cultures of NIT-1 cells, EUK-8 inhibited cytotoxicity caused by superoxide as well as nitric oxide. Collectively, our findings implicate a greater role for nitrogen oxides (other than peroxynitrite) in β-cell damage. Antioxidants designed to prevent the formation of both cytotoxic reactive oxygen and nitrogen species may effectively protect β-cells from spontaneous autoimmunity and alloresponses., The pathology of type 1 diabetes is characterized by the infiltration of the pancreatic islets by T-and B-cells and macrophages. These cells mediate β-cell destruction by the production of proinflammatory [...]

S2 is 30-fold more water-soluble, over 10-fold more potent in inhibition of platelet aggregation, and more effectively reduces ROS generation, protects primary neuronal cells from OGD/R-induced damage, as well as improves PK profile and active component distribution in rodent brain as compared to 3-n-butylphthalide (NBP), suggesting that S2may be a potential anti-ischemia stroke agent.

Piperlongumine 1increases reactive oxygen species (ROS) levels and preferably induces cancer cell apoptosis by triggering different pathways. However, the poor solubility of 1limits its intensive investigation and clinical application. Ligustrazine possesses a water-soluble pyrazine skeleton and can inhibit proliferation and metastasis of cancer cells. We synthesized compound 3by replacement of the trimethoxyphenyl of 1with ligustrazine moiety and further introduced 2-Cl, -Br, and -I to 3for synthesis of 4–6, respectively. Compound 4possessed 14-fold greater aqueous solubility than 1and increased ROS levels in colorectal cancer HCT-116 cells. Additionally, 4preferably inhibited proliferation, migration, invasion, and heteroadhesion of HCT-116 cells. Treatment with 4suppressed tumor growth and lung metastasis in vivo and prolonged the survival of tumor-bearing mice. Furthermore, 4mitigated TGF-β1-induced epithelial-mesenchymal transition and Wnt/β-catenin activation by inhibiting the Akt and GSK-3β phosphorylation in HCT-116 cells. Collectively, 4displayed significant antiproliferation and antimetastasis activities, superior to 1.

Novel furoxan-based nitric oxide (NO) releasing derivatives (8a–p) of farnesylthiosalicylic acid (FTS) were synthesized. Compound 8ldisplayed the strongest inhibition on the proliferation of human hepatocellular carcinoma (HCC) cells in vitro, superior to FTS, sorafenib, and furoxan moiety, selectively induced high frequency of HCC cell apoptosis, and produced high levels of NO in HCC cells but not in nontumor liver cells. Furthermore, 8lexhibited low acute toxicity to mice and significantly inhibited the growth of HCC tumors in vivo and the Ras-related signaling in the tumors. Therefore, our novel findings may provide a new framework for the design of new NO-releasing furoxan/FTS hybrids for the intervention of human HCC.

Currently, no effective and safe medicines are available to treat diabetic nephropathy (DN). Bardoxolone methyl (CDDO-Me) has displayed promising anti-DN activity as well as serious side effects in clinical trials, probably because the highly reactive α-cyano-α,β-unsaturated ketone (CUK) in ring A of CDDO-Me can covalently bind to thiol functionalities in many biomacromolecules. In this study, we designed and synthesized a γ-glutamyl transpeptidase (GGT)-based and CUK-modified derivative of CDDO-Me (2) to address this issue. 2can be specifically cleaved by GGT, which is highly expressed in the kidney, to liberate CDDO-Me in situ. It should be noted that 2exhibited anti-DN efficacy comparable to that of CDDO-Me with much less toxicity in cells and db/dbmice, suggesting that its safety is better than CDDO-Me. Our findings not only reveal the therapeutic potential of 2but also provide a strategy to optimize other synthetic molecules or natural products bearing a pharmacophore like CUK to achieve safer pharmaceutical drugs.

The analgesic and addictive properties of morphine and other opioid drugs are thought to result from their interaction with μ opioid receptors. Using a δ opioid receptor cDNA as a probe, we have isolated a murine μ opioid receptor cDNA clone (mMOR). Stable expression of mMOR in Chinese hamster ovary cells conferred high binding affinity for μ receptor ligands including morphine and [D-Ala2,N-methyl-Phe4,Gly5-ol]-enkephalin and low affinity for δ and κ preferring ligands. Treatment of these cell lines with morphine and other μ agonists inhibited forskolin-induced cAMP accumulation, demonstrating a functional coupling of mMOR to the inhibition of adenylate cyclase. The predicted amino acid sequence of mMOR shares ≈55% overall amino acid identity with the δ receptor and ≈97% identity with the recently reported rat μ opioid receptor. Expression of the μ receptor in mouse brain as revealed by in situhybridization parallels the reported pattern of distribution of μ-selective ligand binding sites. Chromosomal localization (to mouse chromosome 10) and Southern analysis are consistent with a single μ opioid receptor gene in the mouse genome, suggesting that the various pharmacologically distinct forms of the μ receptor arise from alternative splicing, post-translational events, or from a highly divergent gene(s).

In nonobese diabetic (NOD) mice, β–cell reactive T–helper type 1 (Th1) responses develop spontaneously and gradually spread, creating a cascade of responses that ultimately destroys the β–cells. The diversity of the autoreactive T–cell repertoire creates a major obstacle to the development of therapeutics. We show that even in the presence of established Th1 responses, it is possible to induce autoantigen–specific anti–inflammatory Th2 responses. Immune deviation of T–cell responses to the β–cell autoantigen glutamate decarboxylase (GAD65), induced an active form of self–tolerance that was associated with an inhibition of disease progression in prediabetic mice and prolonged survival of syngeneic islet grafts in diabetic NOD mice. Thus, modulation of autoantigen–specific Th1/Th2 balances may provide a minimally invasive means of downregulating established pathogenic autoimmune responses.

The nature (Th1 versus Th2) and dynamics of the autoimmune response during the development of insulin-dependent diabetes mellitus (IDDM) and after immunotherapy are unclear. Here, we show in nonobese diabetic (NOD) mice that the autoreactive T cell response starts and spreads as a pure Th1 type autoimmunity, suggesting that a spontaneous Th1 cascade underlies disease progression. Surprisingly, induction of antiinflammatory Th2 responses to a single β cell antigen (βCA) resulted in the spreading of Th2 cellular and humoral immunity to unrelated βCAs in an infectious manner and protection from IDDM. The data suggest that both Th1 and Th2 autoimmunity evolve in amplificatory cascades by generating site-specific, but not antigen-specific, positive feedback circuits. Determinant spreading of Th2 responses may be a fundamental mechanism underlying antigen-based immunotherapeutics, explaining observations of infectious tolerance and providing a new theoretical framework for therapeutic intervention.

The treatment of ischemic stroke (IS) remains a big challenge in clinics, and it is urgently needed to develop novel, safe, and effective medicines against IS. Here, we report the design, synthesis, and biological evaluation of organic NO2–donors as potential agents for the treatment of IS. The representative compound 4awas able to slowly generate low concentrations of NO2–by reaction with a thiol-containing nucleophile, and the NO2–was selectively converted into NO under ischemic/hypoxia conditions to protect primary rat neurons from oxygen–glucose deprivation and recovery (OGD/R)-induced cytotoxicity by enhancing the Nrf2 signaling and activating the NO/cGMP/PKG pathway. Treatment with 4aat 2 h before or after ischemia mitigated the ischemia/reperfusion-induced brain injury in middle cerebral artery occlusion (MCAO) rats by producing NO and enhancing Nrf2 signaling. Furthermore, 4asignificantly promoted endothelial cell proliferation and angiogenesis within the ischemic penumbra. Our findings suggest that this type of NO2–donors, like 4a, may be valuable to fight IS and other ischemic diseases.