In the nonobese diabetic (NOD) mouse model of type 1 diabetes (T1D), an insulin peptide (B:9-23) is a major target for pathogenic CD4 + T cells. However, there is no consensus on the relative importance of the various positions or "registers" this peptide can take when bound in the groove of the NOD MHCII molecule, IA g7 . This has hindered structural studies and the tracking of the relevant T cells in vivo with fluorescent peptide-MHCII tetramers. Using mutated B:9-23 peptides and methods for trapping the peptide in particular registers, we show that most, if not all, NOD CD4 + T cells react to B:9-23 bound in low-affinity register 3. However, these T cells can be divided into two types depending on whether their response is improved or inhibited by substituting a glycine for the B:21 glutamic acid at the p8 position of the peptide. On the basis of these findings, we constructed a set of fluorescent insulin-IA g7 tetramers that bind to most insulin-specific Tcell clones tested. A mixture of these tetramers detected a high frequency of B:9-23-reactive CD4 + T cells in the pancreases of prediabetic NOD mice. Our data are consistent with the idea that, within the pancreas, unique processing of insulin generates truncated peptides that lack or contain the B:21 glutamic acid. In the thymus, the absence of this type of processing combined with the low affinity of B:9-23 binding to IA g7 in register 3 may explain the escape of insulin-specific CD4 + T cells from the mechanisms that usually eliminate self-reactive T cells.antigen processing | autoimmunity | T cell receptor | self tolerance I n human type 1 diabetes (T1D) and in the nonobese diabetic (NOD) mouse model of the disease, insulin is a major autoantigen for both B cells and T cells (reviewed in refs. 1, 2). A peptide from the insulin beta chain (B:9-23) has been known for many years to be the major target of insulin-reactive CD4 + T cells in NOD T1D . However, the data suggest that this peptide can bind to the NOD class II major histocompatibility (MHCII), IA g7 , in multiple positions or "registers" within the peptide binding groove (3-7). These registers are defined by the peptide amino acids occupying positions p1-p9 in the groove, which include the "anchor" amino acids at p1, p4, p6, and p9, whose side chains interact with compatible pockets in the MHC groove (8, 9). For an individual peptide, each shift in register puts a new set of peptide amino acids into these anchor positions and brings a different set of peptide amino acid side chains to the surface for potential T-cell recognition, generating a unique ligand. Defining which of the possible B:9-23 binding register(s) in the IA g7 groove create the ligand(s) for diabetogenic insulin-reactive T cells has been difficult, leading to uncertainty in exactly how this peptide is processed and presented to T cells in the pancreas and the inability to construct the relevant fluorescent insulin-IA g7 multimers for in vivo tracking the autoimmune B:9-23-specific T cells.Recently, using techniques to trap versio...

Photobiomodulation with near infrared light (NIR) provides cellular protection in various disease models. Previously, infrared light emitted by a low-energy laser has been shown to significantly improve recovery from ischemic injury of the canine heart. The goal of this investigation was to test the hypothesis that NIR (670 nm) from light emitting diodes produces cellular protection against hypoxia and reoxygenation-induced cardiomyocyte injury. Additionally, nitric oxide (NO) was investigated as a potential cellular mediator of NIR. Our results demonstrate that exposure to NIR at the time of reoxygenation protects neonatal rat cardiomyocytes and HL-1 cells from injury, as assessed by lactate dehydrogenase release and MTT assay. Similarly, indices of apoptosis, including caspase 3 activity, annexin binding and the release of cytochrome c from mitochondria into the cytosol, were decreased after NIR treatment. NIR increased NO in cardiomyocytes, and the protective effect of NIR was completely reversed by the NO scavengers carboxy-PTIO and oxyhemoglobin, but only partially blocked by the NO synthase (NOS) inhibitor L-NMMA. Mitochondrial metabolism, measured by ATP synthase activity, was increased by NIR, and NO-induced inhibition of oxygen consumption with substrates for complex I or complex IV was reversed by exposure to NIR. Taken together these data provide evidence for protection against hypoxia and reoxygenation injury in cardiomyocytes by NIR in a manner that is dependent upon NO derived from NOS and non-NOS sources.

Background-Nitric oxide is known to be essential for early anesthetic (APC) and ischemic (IPC) preconditioning of myocardium. Heat shock protein 90 (Hsp90) regulates endothelial nitric oxide synthase (eNOS) activity. In this study, we tested the hypothesis that Hsp90-eNOS interactions modulate APC and IPC.

Backround Reactive oxygen species (ROS) mediate the effects of anesthetic precondition to protect against ischemia and reperfusion injury, but the mechanisms of ROS generation remain unclear. In this study, we investigated if mitochondria-targeted antioxidant (mitotempol) abolishes the cardioprotective effects of anesthetic preconditioning. Further, we investigated the mechanism by which isoflurane alters ROS generation in isolated mitochondria and submitochondrial particles. Methods Rats were pretreated with 0.9% saline, 3.0 mg/kg mitotempol in the absence or presence of 30 min exposure to isoflurane. Myocardial infarction was induced by left anterior descending artery occlusion for 30 min followed by reperfusion for 2h and infarct size measurements. Mitochondrial ROS production was determined spectrofluorometrically. The effect of isoflurane on enzymatic activity of mitochondrial respiratory complexes was also determined. Results Isoflurane reduced myocardial infarct size (40±9 % = mean±SD) compared to control experiments (60±4 %). Mitotempol abolished the cardioprotective effects of anesthetic preconditioning (60±9%). Isoflurane enhanced ROS generation in submitochondrial particles with NADH, but not with succinate, as substrate. In intact mitochondria, isoflurane enhanced ROS production in the presence of rotenone, antimycin A, or ubiquinone when pyruvate and malate were substrates, but isoflurane attenuated ROS production when succinate was substrate. Mitochondrial respiratory experiments and electron transport chain complex assays revealed that isoflurane inhibited only complex I activity. Conclusions The results demonstrated that isoflurane produces ROS at complex I and III of the respiratory chain via the attenuation of complex I activity. The action on complex I decreases unfavorable reverse electron flow and ROS release in myocardium during reperfusion.

The development of an acute infection model of Mycobacterium intracellulare disease in Beige mice (C57B1/6J-bgJ/bgJ) was investigated. Intravenous injection of 1 x 10(7) viable colony-forming units of M. intracellulare strain 571-8 produced a 1,000-fold increase in the number of mycobacteria recovered from the lungs and spleens after 28 days of infection. More importantly, 40% of the beige mice died from this infection, as was determined from histopathologic sections and bacterial culturing.

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