LOX-1, a lectin-like 52-kD receptor for oxidized low-density lipoproteins (ox-LDL), is present primarily on endothelial cells. This receptor is upregulated by ox-LDL itself and by angiotensin II, endothelin, cytokines, and shear stress, all participants in atherosclerosis. This receptor is upregulated in the arteries of hypertensive, dyslipidemic, and diabetic animals. Upregulation of LOX-1 has been identified in atherosclerotic arteries of several animal species and humans, not only on the endothelial lining, but also in the neovasculature of the atherosclerotic plaque, and this receptor is often co-localized with apoptotic cells. Recent studies show upregulation of LOX-1 in the ischemic-reperfused myocardium. LOX-1 inhibition is associated with attenuation of atherosclerosis and associated ischemic injury. LOX-1 may be a novel, exciting target for drug therapy.

Abstract-Atherosclerosis is associated with oxidative stress and inflammation, and upregulation of LOX-1, an endothelial receptor for oxidized LDL (oxLDL). Here, we describe generation of LOX-1 knockout (KO) mice in which binding of oxLDL to aortic endothelium was reduced and endothelium-dependent vasorelaxation preserved after treatment with oxLDL (PϽ0.01 versus wild-type mice). To address whether endothelial functional preservation might lead to reduction in atherogenesis, we crossed LOX-1 KO mice with LDLR KO mice and fed these mice 4% cholesterol/10% cocoa butter diet for 18 weeks. Atherosclerosis was found to cover 61Ϯ2% of aorta in the LDLR KO mice, but only 36Ϯ3% of aorta in the double KO mice. Luminal obstruction and intima thickness were significantly reduced in the double KO mice (versus LDLR KO mice). Expression of redox-sensitive NF-B and the inflammatory marker CD68 in LDLR KO mice was increased (PϽ0.01 versus wild-type mice), but not in the double KO mice. On the other hand, antiinflammatory cytokine IL-10 expression and superoxide dismutase activity were low in the LDLR KO mice (PϽ0.01 versus wild-type mice), but not in the double KO mice. Endothelial nitric oxide synthase expression was also preserved in the double KO mice. The proinflammatory signal MAPK P38 was activated in the LDLR KO mice, and LOX-1 deletion reduced this signal.

Adeno-associated virus (AAV) is a human DNA virus that has a broad host range and can be grown both as an integrated provirus and as a lytic genome. These (10)(11)(12). When no helper virus is available, AAV can persist as an integrated provirus (2, 13-16). Integration apparently involves recombination between AAV termini and host sequences and most of the AAV sequences remain intact (15,16) in the provirus. The ability of AAV to integrate into host DNA is apparently a strategy for insuring the survival of AAV sequences in the absence of the helper virus (16). When cells carrying an AAV provirus are subsequently superinfected with a helper, the integrated AAV genome is rescued and a productive lytic cycle occurs (2).Much of the recent genetic work with AAV (17-19) has been facilitated by the discovery that AAV sequences that have been cloned into prokaryotic plasmids are infectious (17). When the wild-type (wt) AAV/pBR322 plasmid pSM620 is transfected into human cells in the presence of adenovirus, the AAV sequences are rescued from the plasmid and a normal AAV lytic cycle ensues (17). This means that it is possible to modify the AAV sequences in the recombinant plasmid and then to grow a viral stock of the mutant by transfecting the plasmid into human cells (18,19).Using this approach, we have demonstrated the existence of at least three phenotypically distinct regions (Fig. 1) in AAV (19). The rep region codes for one or more proteins that are required for DNA replication and for rescue from the recomThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. binant plasmid. The cap and lip regions (Fig. 1) appear to code for AAV capsid proteins and mutants within these regions are capable of DNA replication (19). In addition, studies of terminal mutants show that the AAV termini are required for DNA replication (18).In this study, we isolate a recombinant AAV viral stock that carries a dominant selectable marker. Using this stock, we demonstrate that AAV can be used to introduce foreign DNA into mammalian cells. Furthermore, we show that when the transduced cells are superinfected with adenovirus, the integrated recombinant genome can be rescued and amplified. These experiments suggest that AAV may be useful as a generalized transduction vector with the unique property that the transduced sequences can be easily recovered from infected cells. MATERIALS AND METHODS

We constructed insertion and deletion mutants with mutations within the adeno-associated virus (AAV) sequences of the infectious recombinant plasmid pSM620. Studies of these mutants revealed at least three AAV phenotypes. Mutants with mutations between 11 and 42 map units were partially or completely defective for rescue and replication of the AAV sequences from the recombinant plasmids (rep mutants). The mutants could be complemented by mutants with replication-positive phenotypes. The protein(s) that is affected in rep mutants has not been identified, but the existence of the rep mutants proves that at least one AAV-coded protein is required for viral DNA replication. Also, the fact that one of the rep mutant mutations maps within the AAV intron suggests that the intron sequences code for part of a functional AAV protein. Mutants with mutations between 63 and 91 map units synthesized normal amounts of AAV duplex DNA but could not generate singlestranded virion DNA (cap mutants). The cap phenotype could be complemented by rep mutants and is probably due to a defect in the major AAV capsid protein, VP3. This suggests that a preformed capsid or precursor is required for the accumulation of single-stranded AAV progeny DNA. Mutants with mutations between 48 and 55 map units synthesized normal amounts of AAV single-stranded and duplex DNA but produced substantially lower yields of infectious virus particles than wild-type AAV (lip mutants). The lip phenotype is probably due to a defect in the minor capsid protein, VP1, and suggests the existence of an additional (as yet undiscovered) AAV mRNA. Evidence is also presented for recombination between mutant AAV genomes during lytic growth.

The defective human parvovirus, adeno-associated virus (AAV), requires multiple functions provided by a coinfecting helper virus for viral replication. In addition, it has recently been shown that at least one AAV gene is also required for AAV DNA replication. In this paper, we investigate the autoregulation of the AAV genome by analyzing the expression of mutant AAV genomes upon transfection into adenovirus-infected human cells. Evidence is presented which indicates that the AAV genome regulates its own gene expression in at least two ways. First, either the AAV p5 gene or both the p5 and p19 genes appear to encode a trans activator of AAV transcription. Frameshift mutations within the p5 or p19 gene severely inhibited the synthesis and accumulation of all AAV transcripts. The defective accumulation of transcripts could be complemented in trans, in a manner independent of DNA replication, by cotransfection with a capsid deletion mutant. Second, evidence is presented which suggests that the p5 and p19 genes contain negative cis-active regulatory elements. Deletion of sequences within the p5 and p19, genes enhanced the accumulation of the p5 transcript in cis upon complementation with an AAV capsid deletion mutant, whereas certain deletions enhanced p40 RNA accumulation in the absence of trans activation by the p5 gene.

We used two kinds of adeno-associated virus (AAV) vectors to transduce the neomycin resistance gene into human cells. The first of these (d152-91) retains the AAV rep genes; the second (d13-94) retains only the AAV terminal repeats and the AAV polyadenylation signal (428 base pairs). Both vectors could be packaged into AAV virions and produced proviral structures that were essentially the same. Thus, the AAV sequences that are required in cis for packaging (pac), integration (int), rescue (res), and replication (ori) of viral DNA are located within a 284-base-pair sequence that includes the terminal repeat. Most of the G418r cell lines (73%)

Interleukin-10 (IL-10) is widely known as an immunosuppressive cytokine by virtue of its ability to inhibit macrophage-dependent antigen presentation, T-cell proliferation, and Th1 cytokine secretion. However, several studies have challenged the perception of IL-10 solely as an immunosuppressive cytokine. As part of an investigation on potentiation of the cytotoxic activity of human papillomavirus E7-specific CD8 ؉ cytotoxic T lymphocytes (CTL) for adoptive transfusions to cervical cancer patients, we found that IL-10 in combination with IL-2, unlike several other combinations, including IL-2 with IL-12, gamma interferon (IFN-␥), tumor necrosis factor alpha, and transforming growth factor ␤, was able to consistently increase cytotoxicity. This augmentation in cytotoxic activity correlated with a significant increase in the cytoplasmic accumulation of perforin as detected by fluorescence-activated cell sorter. Surface expression of both the ␣ and ␤ chains of the CD8 heterodimeric coreceptor and CD56 molecules was increased by exposure of CTL to IL-10. More importantly, we found that administration of IL-10 in combination with IL-2 after antigen stimulation consistently increased the intracellular expression of Th1 cytokines (i.e., IFN-␥ and IL-2) compared to results for control CD8 ؉ T cells cultured in IL-2 alone. In kinetic studies, proliferation, intracellular perforin levels, cytotoxic activity, and IFN-␥ expression were consistently elevated in CTL cultures containing IL-10 compared to control cultures, both at early and late time points following stimulation. In contrast, intracellular IL-2 expression was consistently increased only at early time points following stimulation with autologous tumor cells or solidphase anti-CD3 antibody. Taken together, these data support the use of IL-10 in combination with IL-2 for the in vitro expansion and potentiation of tumor-specific CTL for clinical use in the therapy of cancer.

We have shown that the pulsing of dendritic cells ( DCs ) with human papillomavirus type 16 ( HPV -16 ) antigen proteins by lipofection stimulates class I -restricted cytotoxic T lymphocyte ( CTL ) response against primary cervical cancer cells. Also, we have shown that adeno -associated virus ( AAV ) was able to effectively deliver a cytokine gene into DCs. It has been our hypothesis that the delivery of antigen genes into DCs, resulting in endogenous and continuous antigen protein expression, may result in an improvement in T -cell priming by DCs. Here, DCs are pulsed ( infected ) with an AAV vector containing the HPV -16 E6 gene. After infection, transduced E6 gene mRNA expression and vector chromosomal integration could be identified in infected DCs. Furthermore, priming rosettes formed at early times when the AAV / E6 vector was used. Most importantly, AAV / E6 vector pulsing of DCs induced, after only 7 days of priming, a strong CTL response against primary cervical cancer cell lines, compared to bacterial E6 protein lipofection. Killing was significantly blocked by the addition of anti -MHC class I antibodies. Fluorescence -activated cell sorter ( FACS ) analysis of resulting primed cell populations revealed higher levels of CD8 + T cells by AAV -based pulsing, with little evidence of CD56 ( NK ). FACS analysis of the DC populations revealed that AAV / E6 vector -pulsed DCs had higher levels of CD80 and lower levels of CD86 than protein -pulsed DCs. These data suggest that rAAV may be appropriate for antigen pulsing of DCs for immunotherapy protocols. Finally, our protocol represents an advance in regards to the time needed for generating a CTL response compared to other techniques. Cancer Gene Therapy ( 2001 ) 8, 948 -957