No abstract

Traumatic injury to the spinal cord initiates a cascade of inflammatory-mediated injury and repair processes within the nervous system. In parallel, spinal injury could influence peripheral mechanisms of host defense (e.g., wound healing, antibody production) by altering lymphocyte phenotype and function. The goal of this study was to evaluate the physiological impact of spinal contusion injury on phenotypic and functional indices of lymphocyte activation. A flow cytometric time-course analysis of lymphocytes isolated from lymph node and spleen revealed an increase in CD4+ and a decrease in CD8+ lymphocytes during the first week post injury. The functional potential of lymphocytes was also evaluated based on their ability to proliferate in the presence of a biologically relevant antigen (myelin basic protein, MBP) or a lymphocyte mitogen. The data revealed increased proliferation to MBP by 3 days postinjury in lymphocytes isolated from lymph node but not spleen. By 1 week postinjury, increased proliferation to mitogen was noted in both the lymph node and the spleen suggesting a general increase in lymphocyte reactivity during this time interval. Circulating corticosterone (CORT), an endogenous glucocorticoid with significant effects on lymphocyte phenotype and function, was elevated within 24 h after spinal cord injury (SCI) and remained above control levels throughout the duration of our studies (up to 1 month postinjury). The present data suggest injury-associated changes in immune cell phenotype and function paralleled by the activation of the hypothalamic-pituitary-adrenal (HPA) axis.

The B7:CD28/CTLA-4 costimulatory pathway plays a critical role in regulating the immune response and thus provides an ideal target for therapeutic manipulation of autoimmune disease. Previous studies have shown that blockade of CD28 signaling by mAbs can both prevent and exacerbate experimental autoimmune encephalomyelitis (EAE). In this study, we have designed two CD28 peptide mimics that selectively block B7:CD28 interactions. By surface plasmon resonance, both the end group-blocked CD28 peptide (EL-CD28) and its retro-inverso isomer (RI-CD28) compete effectively with the extracellular domain of CD28 for binding to B7-1. Both the CD28 peptide mimics inhibited expansion of encephalitogenic T cells in vitro. A single administration of EL-CD28 or RI-CD28 peptide significantly reduced disease severity in EAE. Importantly, we show that either CD28 peptide mimic administered during acute disease dramatically improved clinical signs of EAE, suppressing ongoing disease. The ratio of CD80:CD86 expression was significantly lower on CD4+ and F4/80+ spleen cells in CD28 peptide-treated mice. Peripheral deletion of Ag-specific CD4+ T cells occurs following in vivo blockade of CD28 with synthetic CD28 peptides.

Complete activation of T cells requires two signals: an Ag-specific signal delivered via the TCR by the peptide-MHC complex and a second costimulatory signal largely provided by B7:CD28/CTLA-4 interactions. Previous studies have shown that B7 blockade can either ameliorate experimental autoimmune encephalomyelitis by interfering with CD28 signaling or exacerbate the disease by concomitant blockade of CTLA-4 interaction. Therefore, we developed a functional CD28 mimic to selectively block B7:CD28 interactions. The design, synthesis, and structural and functional properties of the CD28 free peptide, the end group-blocked CD28 peptide, and its retro-inverso isomer are shown. The synthetic T cell-costimulatory receptor peptides fold into a polyproline type II helical structure commonly seen in regions of globular proteins involved in transient protein-protein interactions. The binding determinants of CD28 can be transferred onto a short peptide mimic of its ligand-binding region. The CD28 peptide mimics effectively block the expansion of encephalitogenic T cells in vitro suggesting the potential usefulness of the peptides for the treatment of autoimmune disease conditions requiring down-regulation of T cell responses.

The effects of the benzodiazepine receptor partial inverse agonist beta-carboline FG 7142 on cortical ACh efflux were determined using in vivo microdialysis in freely-moving rats. Additionally, a within-subjects, repeated-dialysis experimental design (four microdialysis sessions; removable dialysis probe) was evaluated as a method for measuring changes in basal and FG 7142-stimulated ACh efflux in the frontoparietal cortex. FG 7142 (4.0, 8.0, and 16.0 mg/kg) produced a 150-470% increase in cortical ACh efflux, with a dose-dependent effect on the duration of the increase in efflux. Basal cortical ACh efflux was lower in session 4 than in session 1. However, the ability of FG 7142 to stimulate efflux was unchanged by repeated dialysis testing. The ability of tetrodotoxin (1.0 microM) to suppress both basal and FG 7142-stimulated ACh efflux was also unaffected by repeated dialysis testing. These results demonstrate that systemically administered benzodiazepine receptor inverse agonists stimulate cortical ACh efflux, and that repeated-measures experimental designs can be valid for determining certain changes in cortical ACh efflux with in vivo microdialysis.