This article is available online at http://www.jlr.org pigmentosa and age-related macular degeneration ( 1-3 ). Very few effective therapies exist for this heterogeneous group of diseases because of the complex pathophysiology associated with genetic and environmental factors. Researchers are looking for cellular second messengers involved in the process of cell death which can be targeted for therapies. The sphingolipid metabolite ceramide is a deadly second messenger in the cell and can induce apoptosis through various mechanisms ( 4 ). Recently, increases in ceramide levels have been shown to be associated with photoreceptor and retinal pigment epithelium (RPE) cell death ( 5-8 ). Here we investigated whether ceramide is involved in photoreceptor cell death in light-induced retinal degeneration (LIRD).LIRD is a useful model for studying the mechanism of photoreceptor cell death because intense light exposure induces oxidative stress-mediated apoptosis of photoreceptor cells and causes retinal degeneration ( 3, 9 ). Since its development in 1966 by Noell et al. ( 10 ), this model, in combination with genetic knock-out models (1)(2)(3)(11)(12)(13)(14), has been extensively used to discover many fundamental mechanisms of photoreceptor function and to test various neuroprotective compounds ( 15-21 ).However, the precise mechanism of light-induced retinal degeneration is currently unclear ( 3, 13 ). In past decades, accumulating evidence suggested that ceramide, the core lipid of sphingolipid metabolism, is a key factor in apoptotic cell death ( 4,(22)(23)(24)(25). Various external stressors, such as hypoxia, chemotherapeutic agents, heat, ultravioAbstract Light-induced retinal degeneration (LIRD) in albino rats causes apoptotic photoreceptor cell death. Ceramide is a second messenger for apoptosis. We tested whether increases in ceramide mediate photoreceptor apoptosis in LIRD and if inhibition of ceramide synthesis protects the retina. Sprague-Dawley rats were exposed to 2,700 lux white light for 6 h, and the retinal levels of ceramide and its intermediary metabolites were measured by GC-MS or electrospray ionization tandem mass spectrometry. Enzymes of the de novo biosynthetic and sphingomyelinase pathways of ceramide generation were assayed, and gene expression was measured. The dosage and temporal effect of the ceramide synthase inhibitor FTY720 on the LIRD retina were measured by histological and functional analyses. Retinal ceramide levels increased coincident with the increase of dihydroceramide at various time points after light stress. Light stress in retina induces ceramide generation predominantly through the de novo pathway, which was prevented by systemic administration of FTY720 (10 mg/kg) leading to the protection of retinal structure and function. The neuroprotection of FTY720 was independent of its immunosuppressive action. We conclude that ceramide increase by de novo biosynthesis mediates photoreceptor apoptosis in the LIRD model and that inhibition of ceramide production protects the retina ag...

Cytotoxic T lymphocytes (CTL) play an important role in the control and elimination of infection by West Nile virus (WNV), yet the class I human leukocyte antigen (HLA)-presented peptide epitopes that enable CTL recognition of WNV-infected cells remain uncharacterized. The goals of this work were first to discover the peptide epitopes that distinguish the class I HLA of WNV-infected cells and then to test the T cell reactivity of newly discovered WNV epitopes. To discover WNV-immune epitopes, class I HLA was harvested from WNV (NY99 strain)-infected and uninfected HeLa cells. Then peptide epitopes were eluted from affinity-purified HLA, and peptide epitopes from infected and uninfected cells were comparatively mapped by mass spectroscopy. Six virus-derived peptides from five different viral proteins (E, NS2b, NS3, NS4b, and NS5) were discovered as unique to HLA-A*0201 of infected cells, demonstrating that the peptides sampled by class I HLA are distributed widely throughout the WNV proteome. When tested with CTL from infected individuals, one dominant WNV target was apparent, two epitopes were subdominant, and three demonstrated little CTL reactivity. Finally, a sequence comparison of these epitopes with the hundreds of viral isolates shows that HLA-A*0201 presents epitopes derived from conserved regions of the virus. Detection and recovery from WNV infection are therefore functions of the ability of class I HLA molecules to reveal conserved WNV epitopes to an intact cellular immune system that subsequently recognizes infected cells.epitope hierarchy ͉ human leukocyte antigen ͉ immunodominance ͉ major histocompatibility complex ͉ mass spectrometry W est Nile virus (WNV) is a single-stranded positive sense RNA flavivirus that is related to other human pathogens such as yellow fever virus and dengue fever virus. In nature, WNV exists in an enzootic cycle between birds and mosquitoes, with other species like horses and humans acting as incidental terminal hosts (1, 2). In humans, WNV causes a nonspecific febrile illness with rare cases of fatal encephalitis (3). Like other flaviviruses, WNV translates its genome into a polyprotein of Ϸ3,400 aa that is proteolytically cleaved into three structural proteins and seven nonstructural proteins (4). In contrast to other RNA viruses, such as HIV and influenza, WNV exhibits a high degree of sequence conservation in its natural reservoir (5). Such sequence conservation makes WNV a promising target with regard to targeting humoral and cellular immune responses to conserved epitopes.WNV elicits a strong immune response from innate and adaptive branches of the immune system (6). With regard to adaptive immunity, WNV infection results in the generation of neutralizing antibodies that can protect mice from lethal infection when given passively (7-9), whereas mice deficient for cytotoxic T lymphocytes (CTL) or class I major histocompatibility complex (MHC) exhibit increased viral burdens and increased mortality (10-13). A key component of the cellular antiviral immune response is ...

Cellular immune mechanisms detect and destroy cancerous and infected cells via the human leukocyte antigen (HLA) class I molecules that present peptides of intracellular origin on the surface of all nucleated cells. The identification of novel, tumor-specific epitopes is a critical step in the development of immunotherapeutics for breast cancer. To directly identify peptide epitopes unique to cancerous cells, secreted human class I HLA molecules (sHLA) were constructed by deletion of the transmembrane and cytoplasmic domain of HLA A*0201. The resulting sHLA-A*0201 was transferred and expressed in breast cancer cell lines MCF-7, MDA-MB-231, and BT-20 as well as in the immortal, nontumorigenic cell line MCF10A. Stable transfectants were seeded into bioreactors for production of > 25 mg of sHLA-A*0201. Peptides eluted from affinity purified sHLA were analyzed by mass spectroscopy. Comparative analysis of HLA-A*0201 peptides revealed 5 previously uncharacterized epitopes uniquely presented on breast cancer cells. These peptides were derived from intracellular proteins with either well-defined or putative roles in breast cancer development and progression: Cyclin Dependent Kinase 2 (Cdk2), Ornithine Decarboxylase (ODC1), Kinetochore Associated 2 (KNTC2 or HEC1), Macrophage Migration Inhibitory Factor (MIF), and Exosome Component 6 (EXOSC6). Cellular recognition of the MIF, KNTC2, EXOSC6, and Cdk2 peptides by circulating CD8+ cells was demonstrated by tetramer staining and IFN-gamma ELISPOT. The identification and characterization of peptides unique to the class I of breast cancer cells provide putative targets for the development of immune diagnostic tools and therapeutics.

Resistance to antibiotics currently used empirically to treat newborns is present in bacteremia-producing E. coli. Clonal spread among newborns of multidrug-resistant E. coli is possible; therefore, continued surveillance is needed. Identification of additional virulence factors associated with increased invasion in neonatal E. coli strains is important and further studies are warranted.

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