Immunotargeting of insulin reactive CD8 T cells to prevent diabetes.

Insulin is one of the earliest targeted autoantigens in the immune destruction of insulin-producing beta cells by autoreactive CD4 and CD8 T cells in type 1 diabetes. In this study, we used Non-obese diabetic (NOD) transgenic T cells engineered to express MHC class I-insulin peptide complexes linked to a T cell activation component (InsCD3-ζ), to target insulin-reactive CD8 T cells. We showed that activated, but not naïve, InsCD3-ζ CD8 T cells killed diabetogenic insulin-reactive CD8 target cells in vitro, inducing antigen-specific cell death mediated via both the release of perforin and the Fas-Fas ligand pathway.

A comparative evaluation of the response to peroxynitrite by a brain endothelial cell line and control of the effects by drug targeting.

The potent oxidant peroxynitrite (ONOO(-)) is formed after the combination of nitric oxide with superoxide and has been closely associated with the pathology of inflammatory disease. In particular, the generation of ONOO(-) has been linked to central nervous system disorders including Alzheimer's and Parkinson's disease, multiple sclerosis and bacterial and viral meningitis. Specifically, ONOO(-) has been implicated in the loss of blood-brain barrier (BBB) integrity during neuroinflammation, but the precise mechanisms through which the molecule acts to mediate neurovascular breakdown have not been established.

Developing a novel model system to target insulin-reactive CD8 T cells.

CD8 T cells play an important role in autoimmune diabetes development, and therefore removing these cells may protect against disease. To test this, we designed a novel method using engineered cells (InsCD3-zeta) to target insulin-specific CD8 T cells. Insulin-reactive target cells were cultured with InsCD3-zeta CD8 T cells and cytotoxicity was assessed.

The acute and chronic phases of chronic relapsing experimental autoimmune encephalomyelitis (CR EAE) are ameliorated by the peroxynitrite decomposition catalyst, 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrinatoiron (III) chloride, (FeTPPS).

There is increasing evidence that the oxidative radical, peroxynitrite (ONOO(-)), is involved in the pathogenesis of inflammatory diseases including multiple sclerosis and the animal counterpart, experimental autoimmune encephalomyelitis (EAE). Compounds that impede the actions of ONOO(-) have proved useful in the control of EAE. In particular, catalytic isomerisation of ONOO(-) to inactive nitrate, through the use of metalloporphyrins, curtails the cellular response to inflammatory stimuli and halts the progression of neuroinflammation during EAE.

Loss of blood-brain barrier integrity in the spinal cord is common to experimental allergic encephalomyelitis in knockout mouse models.

Experimental allergic encephalomyelitis (EAE) is an inflammatory demyelinating disease of the CNS that is used to model certain parameters of multiple sclerosis. To establish the relative contributions of T cell reactivity, the loss of blood-brain barrier (BBB) integrity, CNS inflammation, and lesion formation toward the pathogenesis of EAE, we assessed the incidence of EAE and these parameters in mice lacking NF-kappaB, TNF-alpha, IFN-alphabeta receptors, IFN-gamma receptors, and inducible nitric oxide synthase. Although increased myelin oligodendrocyte glycoprotein-specific T cell reactivity was generally associated with a more rapid onset or increased disease severity, the loss of BBB integrity and cell accumulation in spinal cord tissues was invariably associated with the development of neurological disease signs.

Uric acid protects against secondary damage after spinal cord injury.

Peroxynitrite contributes to the pathogenesis of various neurodegenerative disorders through multiple mechanisms and is thought to mediate secondary neuronal cell death after spinal cord injury (SCI). Here we establish that physiologically relevant levels of uric acid (UA), a selective inhibitor of certain peroxynitrite-mediated reactions, block the toxic effects of peroxynitrite on primary spinal cord neurons in vitro. Furthermore, administration of UA at the onset of SCI in a mouse model inhibits several pathological changes in the spinal cord including general tissue damage, nitrotyrosine formation, lipid peroxidation, activation of poly(ADP-ribose) polymerase, and neutrophil invasion.

Poly(ADP-ribose) polymerase activity contributes to peroxynitrite-induced spinal cord neuronal cell death in vitro.

Peroxynitrite, which has been implicated in secondary neuronal damage resulting from spinal cord injury, is capable of mediating several toxic interactions including inducing DNA strand breaks and activating the nuclear enzyme, poly (ADP-ribose) polymerase (PARP). In the present study we have tested the hypothesis that peroxynitrite-induced cell death in spinal cord injury is due to activation of PARP. Initially we examined whether peroxynitrite exerts toxic effects on primary cultures of spinal cord neurons and then determined whether the spinal cord neuronal cell death triggered by peroxynitrite was associated with PARP activation.

ICAM-1 upregulation in the spinal cords of PLSJL mice with experimental allergic encephalomyelitis is dependent upon TNF-alpha production triggered by the loss of blood-brain barrier integrity.

Urate (UA) selectively scavenges peroxynitrite-dependent radicals and suppresses CNS inflammation through effects that are manifested at the blood-brain barrier (BBB). ICAM-1 upregulation in the spinal cord tissues of myelin basic protein (MBP) immunized mice is selectively inhibited by UA treatment. In contrast, the expression of ICAM-1 and other adhesion molecules by circulating cells is unchanged.

The therapeutic effects of PJ34 [N-(6-oxo-5,6-dihydrophenanthridin-2-yl)-N,N-dimethylacetamide.HCl], a selective inhibitor of poly(ADP-ribose) polymerase, in experimental allergic encephalomyelitis are associated with immunomodulation.

Poly(ADP-ribose) polymerase (PARP) activity has been implicated in the pathogenesis of several central nervous system (CNS) disorders. For example, the presence of extensive poly(ADP)ribosylation in CNS tissues from animals with experimental allergic encephalomyelitis (EAE) indicates that PARP activity may be involved in this inflammatory disease process. Using PJ34 [N-(6-oxo-5,6-dihydrophenanthridin-2-yl)-N, N-dimethylacetamide.

Differential expression of growth factors at the cellular level in virus-infected brain.

The contribution of host factors to rabies virus (RV) transcription/replication and axonal/transsynaptic spread is largely unknown. We previously identified several host genes that are up-regulated in the mouse brain during RV infection, including neuroleukin, which is involved in neuronal growth and survival, cell motility, and differentiation, and fibroblast growth factor homologous factor 4 (FHF4), which has been implicated in limb and nervous system development. In this study, we used real-time quantitative RT-PCR to assess the expression of mRNAs specific for neuroleukin, the two isoforms of FHF4 (FHF4-1a and -1b) encoded by the FHF4 gene, and N protein of RV in neurons and astrocytes isolated by laser capture microdissection from mouse brains infected with the laboratory-adapted RV strain CVS-N2c or with a street RV of silver-haired bat origin.

Peroxynitrite-induced oligodendrocyte toxicity is not dependent on poly(ADP-ribose) polymerase activation.

Oligodendrocyte loss is a characteristic feature of several CNS disorders, including multiple sclerosis (MS) and spinal cord injury. However, the mechanisms responsible for oligodendrocyte destruction remain undefined. As recent studies have implicated peroxynitrite in the pathogenesis of both spinal cord injury and MS, we have examined whether peroxynitrite may mediate at least some of the oligodendrocyte damage and demyelination observed in these conditions.

Therapeutic intervention in experimental allergic encephalomyelitis by administration of uric acid precursors.

Uric acid (UA) is a purine metabolite that selectively inhibits peroxynitrite-mediated reactions implicated in the pathogenesis of multiple sclerosis (MS) and other neurodegenerative diseases. Serum UA levels are inversely associated with the incidence of MS in humans because MS patients have low serum UA levels and individuals with hyperuricemia (gout) rarely develop the disease. Moreover, the administration of UA is therapeutic in experimental allergic encephalomyelitis (EAE), an animal model of MS.

Comparison of uric acid and ascorbic acid in protection against EAE.

Serum levels of uric acid (UA), an inhibitor of peroxynitrite- (ONOO-) related chemical reactions, became elevated approximately 30 million years ago in hominid evolution. During a similar time frame, higher mammals lost the ability to synthesize another important radical scavenger, ascorbic acid (AA), leading to the suggestion that UA may have replaced AA as an antioxidant. However, in vivo treatment with AA does not protect against the development of experimental allergic encephalomyelitis (EAE), a disease that has been associated with the activity of ONOO- and is inhibited by UA.

Inhibition of hypochlorous acid-induced cellular toxicity by nitrite.

Chronic inflammation results in increased nitrogen monoxide (.NO) formation and the accumulation of nitrite (NO(2-)). Neutrophils stimulated by various inflammatory mediators release myeloperoxidase to produce the cytotoxic agent hypochlorous acid (HOCl).

Circadian variation in oxidative stress markers in healthy and type II diabetic men.

Seven clinically healthy, nondiabetic (ND) and four Type II diabetic (D) men were assessed for circadian rhythms in oxidative "stress markers." Blood samples were collected at 3h intervals for approximately 27 h beginning at 19:00h. Urine samples were collected every 3 h beginning with the 16:00h-19:00h sample.