Grace Ku

Amyloid-β peptide induces oligodendrocyte death by activating the neutral sphingomyelinase-ceramide pathway

Amyloid-β peptide (Aβ) accumulation in senile plaques, a pathological hallmark of Alzheimers disease (AD), has been implicated in neuronal degeneration. We have recently demonstrated that Aβ induced oligodendrocyte (OLG) apoptosis, suggesting a role in white matter pathology in AD. Here, we explore the molecular mechanisms involved in Aβ-induced OLG death, examining the potential role of ceramide, a known apoptogenic mediator. Both Aβ and ceramide induced OLG death. In addition, Aβ activated neutral sphingomyelinase (nSMase), but not acidic sphingomyelinase, resulting in increased ceramide generation. Blocking ceramide degradation with N-oleoyl-ethanolamine exacerbated Aβ cytotoxicity; and addition of bacterial sphingomyelinase (mimicking cellular nSMase activity) induced OLG death. Furthermore, nSMase inhibition by 3-O-methyl-sphingomyelin or by gene knockdown using antisense oligonucleotides attenuated Aβ-induced OLG death. Glutathione (GSH) precursors inhibited Aβ activation of nSMase and prevented OLG death, whereas GSH depletors increased nSMase activity and Aβ-induced death. These results suggest that Aβ induces OLG death by activating the nSMase–ceramide cascade via an oxidative mechanism.

iNOS and nitrotyrosine expression after spinal cord injury

Secondary tissue damage after spinal cord injury (SCI) may be due to inflammatory mediators. After SCI, the nuclear factor-kappaB (NF-kappaB) transcription factor can activate many pro-inflammatory genes, one of which is inducible nitric oxide synthase (iNOS). iNOS catalyzes the synthesis of nitric oxide (NO), a key inflammatory mediator, which in turn reacts with superoxide to generate peroxynitrite. Peroxynitrite is a strong oxidant that can damage cellular enzymes, membranes, and subcellular organelles through the nitration of tyrosine residues on proteins. The presence of nitrotyrosine (NT) is an indirect chemical indicator of toxic NO and peroxynitrite-induced cellular damage. Using a New York University (NYU) impactor to induce SCI in adult rats, we examined the temporal and cellular expression of iNOS and NT. We observed a progressive increase in iNOS expression in the injured cord starting at day 1 with maximal expression occurring at day 7, as determined by Western blot analysis. iNOS expression corresponded temporally to an increase in iNOS enzyme activity after SCI. In parallel with the progressive increase in iNOS activity, NT expression also increased with time after SCI. The iNOS and NT immunoreactivity was localized in neurons, astrocytes, endothelial cells and ependymal cells at the epicenter and adjacent to the region of spinal cord impact and injury. Results from the present study suggest that increased iNOS and peroxynitrite anion, as reflected by the progressive accumulation of NT in the injured impacted spinal cord, may contribute to the secondary injury process after SCI.

Amyloid β Peptide–Induced Cerebral Endothelial Cell Death Involves Mitochondrial Dysfunction and Caspase Activation

Amyloid β peptide (Aβ), a 39 to 43 amino acid fragment of the β-amyloid precursor protein (βAPP), forms insoluble fibrillar accumulation in neurofibrillary tangles and vascular plaques. Aβ has been implicated in neuronal and vascular degeneration in brain regions susceptible to plaque formation because of its cytotoxic effect on neurons and endothelial cells (ECs). The authors used a murine cerebral endothelial cell (CEC) line and primary cultures of bovine CECs to explore the cytotoxic mechanism of Aβ. Aβ 1–40 and Aβ 25–35 peptides caused cell death in a dose-dependent and time-dependent manner. Exposure to either Aβ 25–35 or Aβ 1–40 at 10 μmol/L for 48 hours caused at least 40% cell death. Cerebral endothelial cell death was characterized by nuclear condensation, mitochondrial dysfunction, and nuclear and mitochondrial DNA damage. Aβ 25–35 activated both caspase-8 and caspase-3 in murine CECs. zVAD-fmk, a broad-spectrum caspase inhibitor, prevented Aβ 25–35-induced increase in caspase-3 activity and CEC death. N-acetyl-cysteine, an antioxidant, also prevented Aβ-induced cell death. Together, these findings indicate that Aβ-mediated CEC death is an apoptotic process that is characterized by increased oxidative stress, caspase activation, mitochondrial dysfunction, and nuclear and mitochondrial DNA damage.

Astrocytes and central nervous system endothelial cells do not express B7-1 (CD80) or B7-2 (CD86) immunoreactivity during experimental autoimmune encephalomyelitis

The identity of cell types within the central nervous system (CNS) capable of activating T lymphocytes is a fundamental issue in the understanding of multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE). To become fully activated, a T cell must recognize its antigen and receive co-stimulation, the latter being optimally delivered via B7-1 and/or B7-2 molecules expressed by the antigen presenting cell (APC). There are conflicting reports regarding whether astrocytes or CNS endothelial cells (EC) can act as fully competent APCs. The present studies were performed to determine whether astrocytes or CNS EC express B7-1 or B7-2 immunoreactivity during EAE. No expression of B7-1 or B7-2 by either astrocytes or EC was detected during acute, remitting, relapsing or chronic EAE, whether EAE was induced by active immunization or cell transfer using five different myelin antigens. These results suggest that neither astrocytes nor CNS EC can deliver co-stimulatory signals via B7 molecules in the setting of murine EAE, rendering them incapable of acting as fully competent APCs.

Development of Infectious Tolerance After Donor-Specific Transfusion and Rat Heart Transplantation

Regulatory cells developed after donor-specific transfusion (DST)-induced acceptance of a LEW heart transplanted into a DA rat. Both DST and the cardiac transplant were necessary to generate the regulatory cells. This donor-specific tolerance can then be transferred into a new DA recipient by adoptive transfer of lymphocytes from the DST-treated long term survivor (LTS) in a dose-dependent manner. The effectiveness of tolerance did not diminish over five generations of adoptive transfer, thus supporting its infectious nature. Although both spleen and lymph node cells were equally effective, graft-infiltrating lymphocytes were more potent. A high level of indirect CTL activity and MLC proliferation were observed in lymphocytes from LTS. In vivo tracking of adoptively transferred CFSE-labeled splenocytes from LTS showed equivalent FACS proliferation and a higher percentage of graft-infiltrating lymphocytes 7 days after heart transplantation, compared with adoptively transferred naive splenocytes. Adoptive transfer of CD8+-depleted LTS splenocytes resulted in 100% subsequent LEW allograft acceptance; whereas CD4+ depletion decreased acceptance to 40%, and depletion of both CD4 and CD8 resulted in 0% acceptance. When positively selected CD4+ or CD8+ cells were adoptively transferred, 100% or 62.5% of LEW cardiac allografts survived, respectively. In conclusion, DST alone promotes a donor-specific infectious tolerance of a heart graft that can be adoptively transferred to subsequent naive allograft recipients despite the undiminished in vitro immunological response to donor Ag. Although both CD4+ and CD8+ populations are responsible for the regulatory mechanism in DST-induced tolerance, the CD4+ population appears to dominate.

T cells are the main cell type expressing B7-1 and B7-2 in the central nervous system during acute, relapsing and chronic experimental autoimmune encephalomyelitis.

T cell co‐stimulation through the CD28 receptor on T cells is critical to the induction of experimental autoimmune encephalomyelitis (EAE). In this study, expression of the co‐stimulatory ligands B7‐1 (CD80) and B7‐2 (CD86), as well as the receptors CD28 and CTLA‐4, were quantitated in central nervous system (CNS) tissues from mice at various stages of EAE. Immunohistochemistry and flow cytometry of CNS‐infiltrating cells revealed a high percentage of infiltrating T cells expressing B7‐1 and B7‐2 during acute, chronic and relapsing EAE. Of the infiltrating cells 10 – 20 % were CTLA‐4+, most of which were CD4+ T cells. B7‐1 and B7‐2 expression within the CNS during active EAE might increase the potential for local activation of autoimmune T cells; however, the high level of expression of B7 molecules may also provide a mechanism for the autoregulation of activated CTLA‐4+ T cells.

Effects of endotoxin tolerance on Propionibacterium acnes-primed lipopolysaccharide hepatic injury,

BACKGROUND Prior administration of the Gram-positive bacteria Propionibacterium acnes (PA) results in hypersensitivity and hepatocyte necrosis to a subsequent low dose of lipopolysaccharide (LPS). Endotoxin tolerance has been shown to prevent lethality after ischemia/reperfusion injuries, sepsis, and endotoxic shock. We investigated whether prior induction of LPS tolerance could prevent subsequent PA-priming and LPS-induced death. The levels of known effector cytokines possibly responsible for these changes were measured. METHODS C57BL/6 (B6) mice were given heat-killed PA (0.5 mg/mouse) followed 7 days later by LPS (20 microg/mouse). In parallel experiments, B6 mice were pretreated with a single 20 microg/mouse dose of LPS (lethal dose = 800 microg/mouse) 7 days prior to PA priming. Animal survival, liver and spleen weights, and histology were examined. Cytokine levels of the inflammatory cytokines interferon-alpha, tumor necrosis factor-gamma, interleukin (IL)-6, and IL-12 and the anti-inflammatory cytokines IL-4 and IL-10 were measured by enzyme-linked immunosorbent assay and by reverse-transcription polymerase chain reaction. RESULTS Hepatomegaly, splenomegaly, and hepatocyte necrosis with death developed in all PA-primed B6 mice challenged with LPS. However, 83% of B6 mice given a tolerizing dose of LPS prior to PA survived (P < 0.01) without any increase in liver or spleen weights and without histological evidence of necrosis. Markedly decreased in vivo and in vitro inflammatory (interferon-alpha, tumor necrosis factor-gamma, IL-6, and IL-12) cytokine levels corresponded with survival in the LPS-tolerant mice. Endotoxin tolerance and subsequent survival were also associated with an increase in anti-inflammatory (IL-4 and IL-10) mRNA expression. CONCLUSIONS Lethal PA-primed LPS-induced hepatic injury can be prevented by administering a tolerizing dose of LPS prior to PA-priming. LPS protects the liver by preventing hepatic mononuclear cellular infiltration, reducing the production of the toxic proinflammatory cytokines, and inducing the production of endogenous anti-inflammatory cytokines.

Interleukin‐12 regulates natural killer cell‐dependent Propionibacterium acnes‐primed, lipopolysaccharide‐induced liver injury

Aim:  Interleukin (IL)‐12, produced primarily by macrophage/monocytes, modulates mature T and natural killer (NK) cell functions, including cytotoxicity and cytokine production.