Introduction and Aims: Chronic kidney disease (CKD) is a major risk factor for atherosclerotic cardiovascular diseases. Recently it has been reported that apoptosis of aortic smooth muscle cells (AoSMCs) may relate to the atherosclerosis with plaque formation or calcification. Therefore, in the present study, we examined the effect of indoxyl sulfate (IS), which is thought to be one of uremic toxin, for apoptosis in cultured rat AoSMCs. Methods: The induction of apoptosis was quantitated by assay of the caspase CPP32, which plays a direct role in the execution of cell death. And the activity of SAPK/JNK and P38 MAP kinase, which is known as apoptosis-inducing signal transduction, was assessed by standard immunoblot using phospho-specific antibodies. Results: Twenty five μg/ml of IS, which is compatible with the concentration of IS in the serum of end-stage renal failure patients, induced 3.9±2.7-fold increase in the caspase CPP32 activity responding to serum withdrawal for 12 hours. The blockade of organic anion transporter (OAT) by 0.5mM probenecid (Pb) abolished the effect of IS on the apoptosis in AoSMCs (relative increase in the caspase CPP32: Pb-, IS-; 1±0.1, Pb-, IS+; 2.4±0.2, Pb+, IS-; 0.9±0.1, Pb+, IS+; 1.2±0.1). Indoxyl sulfate activated SAPK/JNK in AoSMCs that was significantly elevated by 30 minutes and sustained for over 2 hours, although it did not affect the activation of P38 MAP kinase. Conclusions: These results indicate that IS accelerates apoptosis induced by serum withdrawal in rat AoSMCs, which is mediated by cellular transport of IS via the OAT and may also be related to the activation of SAPK/JNK pathway. The induction of apoptosis by the accumulation of IS in blood due to CKD may play an important role in atherosclerotic lesion formation.

A new langerin ؉ DC subset has recently been identified in murine dermis (langerin ؉ dDC), but the lineage and functional relationships between these cells and langerin ؉ epidermal Langerhans cells (LC) are incompletely characterized. Selective expression of the cell adhesion molecule EpCAM by LC allowed viable LC to be easily distinguished from langerin ؉ dDC in skin and lymphoid tissue and ex vivo as well. Differential expression of EpCAM and langerin revealed the presence of at least 3 distinct skin DC subsets. We determined that LC and langerin ؉ dDC exhibit different migratory capabilities in vitro and repopulate distinct anatomic compartments in skin at different rates after conditional depletion in vivo. Langerin ؉ dDC, in contrast to LC, did not require TGF␤1 for development. Carefully timed gene gun immunization studies designed to take advantage of the distinct repopulation kinetics of langerin ؉ dDC and LC revealed that langerin ؉ dDC were required for optimal production of ␤-galactosidase-specific IgG2a/c and IgG2b in the acute phase. In contrast, immunization via LC-deficient skin resulted in persistent and strikingly reduced IgG1 and enhanced IgG2a Ab production. Our data support the concepts that LC and langerin ؉ dDC represent distinct DC subsets that have specialized functions and that LC are important immunoregulatory cells. The presence of at least 3 functionally distinct skin DC subsets may have particular relevance for vaccines that are administered epicutaneously.EpCAM ͉ gene gun ͉ langerin ͉ TGF-beta T he remarkable phenotypic heterogeneity of DC, both between and within certain tissues, has been long recognized. To date, however, it has been possible to clearly relate DC phenotype to DC function in only a few instances, even in mice. For example, plasmacytoid DC are recognized as the primary source of virus-induced type I IFN (1), CD8␣ ϩ lymph node DC are largely responsible for cross-presentation of cell-associated antigen to CD8 T cells (2-4), and 33D1-reactive (DCIR2 ϩ ) splenic DC (as compared with CD205 ϩ DC) preferentially stimulate CD4 T cells (5). Epidermal Langerhans cells (LC) represent perhaps the most striking example of an extensively studied tissue DC subpopulation whose function is incompletely understood.LC have long been thought to play pivotal roles in initiating immunity by acquiring antigens that are encountered in skin, migrating to draining LN after activation, and stimulating antigen-specific T cells (6). However, recent studies suggested that LC do not function as essential antigen-presenting cells for anti-viral immune responses (2, 7) or for contact hypersensitivity reactions (8-12) in established murine models. Studies of LC have been challenging, in part, because readily detectable cell surface proteins that are constitutively expressed by epidermal LC and LC that have emigrated from epidermis have not been well recognized. The C-type lectin langerin has been regarded as a pathognomonic LC marker, but recent studies suggest that this protein is present in at le...

'Naked' nucleic acid vaccines are potentially useful candidates for the treatment of patients with cancer, but their clinical efficacy has yet to be demonstrated. We sought to enhance the immunogenicity of a nucleic acid vaccine by making it 'self-replicating'. We accomplished this by using a gene encoding an RNA replicase polyprotein derived from the Semliki forest virus, in combination with a model antigen. A single intramuscular injection of a self-replicating RNA immunogen elicited antigen-specific antibody and CD8+ T-cell responses at doses as low as 0.1 microg. Pre-immunization with a self-replicating RNA vector protected mice from tumor challenge, and therapeutic immunization prolonged the survival of mice with established tumors. The self-replicating RNA vectors did not mediate the production of substantially more model antigen than a conventional DNA vaccine did in vitro. However, the enhanced efficacy in vivo correlated with a caspase-dependent apoptotic death in transfected cells. This death facilitated the uptake of apoptotic cells by dendritic cells, providing a potential mechanism for enhanced immunogenicity. Naked, non-infectious, self-replicating RNA may be an excellent candidate for the development of new cancer vaccines.

DNA vaccines were introduced less than a decade ago but have already been applied to a wide range of infectious and malignant diseases. Here we review the current understanding of the mechanisms underlying the activities of these new vaccines. We focus on recent strategies designed to enhance their function including the use of immunostimulatory (CpG) sequences, dendritic cells (DC), costimulatory molecules and cytokine-and chemokine-adjuvants. Although genetic vaccines have been significantly improved, they may not be sufficiently immunogenic for the therapeutic vaccination of patients with infectious diseases or cancer in clinical trials. One promising approach aimed at dramatically increasing the immunogenicity of genetic vaccines involves making them 'selfreplicating'. This can be accomplished by using a gene encoding RNA replicase, a polyprotein derived from alphaviruses, such as Sindbis virus. Replicase-containing RNA vectors are significantly more immunogenic than conventional plasmids, immunizing mice at doses as low as 0.1 μg of nucleic acid injected once intramuscularly. Cells transfected with 'self-replicating' vectors briefly produce large amounts of antigen before undergoing apoptotic death. This death is a likely result of requisite double-stranded (ds) RNA intermediates, which also have been shown to super-activate DC. Thus, the enhanced immunogenicity of 'self-replicating' genetic vaccines may be a result of the production of pro-inflammatory dsRNA, which mimics an RNA-virus infection of host cells.

B-cell functions in antitumor immunity are not well understood. In this study, we evaluated the role of B cells in the development of antitumor immunity using Friend murine leukemia virus gag-expressing mouse EL-4 (EL-4 gag), D5 mouse melanoma, or MCA304 mouse sarcoma cells.

Cancer vaccines targeting 'self' antigens that are expressed at consistently high levels by tumor cells are potentially useful in immunotherapy, but immunological tolerance may block their function. Here, we describe a novel, naked DNA vaccine encoding an alphavirus replicon (self-replicating mRNA) and the self/tumor antigen tyrosinase-related protein-1. Unlike conventional DNA vaccines, this vaccine can break tolerance and provide immunity to melanoma. The vaccine mediates production of double-stranded RNA, as evidenced by the autophosphorylation of protein kinase R. Double-stranded RNA is critical to vaccine function because both the immunogenicity and the antitumor activity of the vaccine are blocked in mice deficient for the RNase L enzyme, a key component of the 2′,5′-linked oligoadenylate synthetase antiviral pathway involved in double-stranded RNA recognition. This study shows for the first time that alphaviral replicon-encoding DNA vaccines activate innate immune pathways known to drive antiviral immune responses, and points the way to strategies for improving the efficacy of immunization with naked DNA.Vaccine vectors based on recombinant viruses have been used for many years, but the delivery of target antigens can be accompanied by unwanted side effects. First, preexisting antibodies can neutralize the recombinant virus before it is able to deliver its payload. Second, structural proteins from the virus can dominate T-and B-cell-mediated immune responses, diverting immunity away from the target immunogen 1,2 . Hence, there is a critical need to develop vaccine vectors that are not only highly immunogenic, but also antigenically simple.The simplest of all recombinant vectors, naked plasmid DNA vaccines, have successfully been used in animal models to induce immune responses to many pathogens and model antigens. DNA vaccines are easy to produce, inexpensive and safe 3 , but for some applications insufficiently immunogenic. A variety of attempts have been made to improve DNA vaccines 4 , including the production of pro-apoptotic proteins 5 . Despite these advances, the poor immunogenicity of plasmid DNA remains apparent when attempting to elicit immunity to weak immunogens, such as non-mutated 'self' tumor-associated antigens that are recognized by anti-tumor T cells. One promising new strategy to improve naked DNA vaccines is to express the target antigen under the control of an alphaviral replicase 6,7 with the premise of using the ability of alphavirus to produce large amounts of viral mRNA (refs. 8 ,9 ). In alphavirus-derived DNA and RNA vaccines, the encoded alphaviral replicase-enzyme complex amplifies self-replicating RNA (replicon). In model systems, replicon containing nucleic acid vaccines display therapeutic efficacy at doses several logs lower than those required by conventional DNA vaccines 10-12 . In the current study, we examined whether a replicase-based DNA vaccine encoding a nonmutated self-antigen could be used to break tolerance and prevent B16 melanoma, a goal not previously ...

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Objective MFG-E8 (lactadherin, SED1) is a secreted glycoprotein that has been previously implicated in enhancement of VEGF-dependent angiogenesis. Major sources of MFG-E8 in vivo, and precise mechanisms of MFG-E8 action remain undetermined. The objective of this study was to identify important sources of MFG-E8 in vivo, and further elucidate the role(s) of MFG-E8 in the regulation of angiogenesis. Methods and Results We utilized knockout mice and anti-MFG-E8 antibodies to study MFG-E8 function in vivo. In melanomas and in retinas of mice with oxygen-induced retinopathy, MFG-E8 colocalized with pericytes rather than endothelial cells, and platelet-derived growth factor receptor β (PDGFRβ)+ pericytes/pericyte precursors purified from tumors contained large amounts of MFG-E8 mRNA. Tumor- and retinopathy-associated angiogenesis was diminished in MFG-E8 knockout mice and pericyte coverage of neovessels was reduced. Inhibition of MFG-E8 production by 10T1/2 cells (surrogate pericyte/pericyte precursors) using small interfering (si)RNAs and short hairpin (sh)RNAs, or inhibition of MFG-E8 action with some anti-MFG-E8 antibodies, selectively attenuated migration in vitro. Significantly, the anti-MFG-E8 antibodies that inhibited 10T1/2 cell migration in vitro also inhibited pathologic angiogenesis in vivo. Conclusions These studies strongly implicate MFG-E8 in pericytes/pericyte precursor function, and indicate that MFG-E8-directed therapeutics may merit further development.