Silencing T-bet defines a critical role in the differentiation of autoreactive T lymphocytes.

As a means of developing therapies that target the pathogenic T cells in multiple sclerosis (MS) without compromising the immune system or eliciting systemic side effects, we investigated the use of T-bet-specific antisense oligonucleotides and small interfering RNAs (siRNA) to silence T-bet expression in autoreactive encephalitogenic T cells and evaluated the biological consequences of this suppression in experimental autoimmune encephalomyelitis, a model for MS. The T-bet-specific AS oligonucleotide and siRNA suppressed T-bet expression, IFNgamma production, and STAT1 levels during antigen-specific T cell differentiation. In vitro suppression of T-bet during differentiation of myelin-specific T cells and in vivo administration of a T-bet-specific antisense oligonucleotide or siRNA inhibited disease. T-bet was shown to bind the IFNgamma and STAT1 promoters, but did not regulate the IL-12/STAT4 pathway. Since T-bet regulates IFNgamma production in CD4(+) T cells, but to a lesser extent in most other IFNgamma-producing cells, T-bet may be a target for therapeutics for Th1-mediated diseases.

Peroxisome proliferator-activated receptor alpha agonists as therapy for autoimmune disease.

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor superfamily. PPAR gamma ligands, which include the naturally occurring PG metabolite 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)), as well as thiazolidinediones, have been shown to have anti-inflammatory activity. The PPAR alpha agonists, gemfibrozil, ciprofibrate, and fenofibrate, have an excellent track history as oral agents used to treat hypertriglyceridemia. In the present study, we demonstrate that these PPAR alpha agonists can increase the production of the Th2 cytokine, IL-4, and suppress proliferation by TCR transgenic T cells specific for the myelin basic protein Ac1-11, as well as reduce NO production by microglia. Oral administration of gemfibrozil and fenofibrate inhibited clinical signs of experimental autoimmune encephalomyelitis. More importantly, gemfibrozil was shown to shift the cytokine secretion of human T cell lines by inhibiting IFN-gamma and promoting IL-4 secretion. These results suggest that PPAR alpha agonists such as gemfibrozil and fenofibrate, may be attractive candidates for use in human inflammatory conditions such as multiple sclerosis.

Immunofluorescent labeling of increased calpain expression and neuronal death in the spinal cord of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated mice.

Parkinson's disease (PD) is a movement disorder characterized by rigidity, tremor, and bradykinesia, originating from degeneration of dopaminergic neurons in the substantia nigra (SN), retrorubral area, and locus ceoruleus (LC). Calpain has been implicated in the pathophysiology of neurodegenerative diseases. Since the spinal cord (SC) and brain are integrally connected and calpain is involved in cell death and mitochondrial dysfunction, we hypothesized that SC neurons are also affected in PD. In order to examine this hypothesis, we examined both brain and SC from mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). To identify cells expressing calpain, double immunofluorescent labeling was performed with antibodies specific for calpain and a cell type (OX-42, GFAP, or NeuN). Combined terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) and double immunofluorescent labeling were used to identify death of specific cells in the central nervous system (CNS). There was an increase in calpain expression in microglia, astrocytes, and neurons in the SC of MPTP-treated mice at 1 and 7 days, as compared to controls. TUNEL-positive neurons in the SC and SN showed apoptotic characteristics. These results demonstrated that neuronal death occurred not only in SN but also in the SC of MPTP-treated mice and has provided evidence for a possible calpain-mediated SC neuronal death in MPTP-induced parkinsonism in mice.

Calpain upregulation and neuron death in spinal cord of MPTP-induced parkinsonism in mice.

Parkinson's disease (PD) is a neurodegenerative disorder resulting in slowness, tremors, and imbalance. Treatment of mice with 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP) is one of several models used to mimic PD in humans. Administration of MPTP leads to the production of 1-methyl-4-phenyl-2,3 dihydropyridinium (MPP(+)). MPP(+) is taken up by dopaminergic neurons, causing mitochondrial dysfunction and cell death. Because calpain is involved in neuronal cell death and mitochondrial dysfunction, we examined the level of calpain in neurons in the substantia nigra (SN) and hippocampus of MPTP-treated C57BL/6 mice. Because of the interconnections between spinal cord and upper central nervous system neurons, we examined morphology, calpain activity, and calpain expression in neurons by double immunofluorescence using calpain and neuron marker (NeuN) antibodies. In controls, calpain expression was low in SN, hippocampus, and spinal cord NeuN(+) cells, and the NeuN stain was concentrated around the nucleus. In mice sacrificed 24 h after administration of three 20 mg/kg doses of MPTP, calpain expression was slightly increased in SN and hippocampal neurons and moderately increased in spinal cord neurons. In these animals, the NeuN stain was less concentrated around the nuclear membrane. One week after MPTP treatment, calpain content in NeuN(+) cells was greatly increased in SN, hippocampus, and spinal cord. Morphologically, SN and spinal cord neurons, treated for one week, were necrotic with a granular cytoplasmic NeuN content. Also, MPTP treatment upregulated calpain activity and mRNA level in spinal cord. These data suggest that following MPTP treatment, calpain causes neuronal death in SN as well as in spinal cord.