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Regulating appropriate activation of the immune response in the healthy host despite continual immune surveillance dictates that immune responses must be either self-limiting and therefore negatively regulated following their activation or prevented from developing inappropriately. In the case of antigen-specific T cells, their response is attenuated by several mechanisms, including ligation of CTLA-4 and PD-1. Through the study of the viral OX2 (vOX2) immunoregulator encoded by Kaposi's sarcoma-associated herpesvirus (KSHV), we have identified a T cell-attenuating role both for this protein and for CD200, a cellular orthologue of the viral vOX2 protein. In vitro , antigen-presenting cells (APC) expressing either native vOX2 or CD200 suppressed two functions of cognate antigen-specific T cell clones: gamma interferon (IFN-γ) production and mobilization of CD107a, a cytolytic granule component and measure of target cell killing ability. Mechanistically, vOX2 and CD200 expression on APC suppressed the phosphorylation of ERK1/2 mitogen-activated protein kinase in responding T cells. These data provide the first evidence for a role of both KSHV vOX2 and cellular CD200 in the negative regulation of antigen-specific T cell responses. They suggest that KSHV has evolved to harness the host CD200-based mechanism of attenuation of T cell responses to facilitate virus persistence and dissemination within the infected individual. Moreover, our studies define a new paradigm in immune modulation by viruses: the provision of a negative costimulatory signal to T cells by a virus-encoded orthologue of CD200.

Rhesus monkey rhadinovirus (RRV) is a gamma-2 herpesvirus related to the human Kaposi's sarcoma-associated herpesvirus (KSHV or human herpesvirus 8). This study identified an alternatively spliced gene at the right side of the RRV genome (strain 17577) between open reading frame 75 and the terminal repeat region. Of its eight exons, the first seven encoded up to 12 transmembrane domains, whilst the eighth exon encoded a predicted C-terminal cytoplasmic domain. Structurally and positionally, this RRV gene therefore resembles the K15 gene of KSHV; it was provisionally named RK15 to avoid confusion with other RRV17577 genes. In ectopic expression studies, the 55 kDa RK15 protein isoform activated the JNK and NF-κB pathways, like the 45 kDa KSHV K15-encoded protein isoform. In contrast to K15, which activates angiogenic and inflammatory cytokines such as interleukin (IL)-8, IL-6 and CCL20, the range of cellular transcripts activated by the RRV K15 homologue was much more restricted, but included IL-6, IL-8 and FGF21. These data suggest functional differences between terminal membrane proteins at the right end of the genomes of Old World primate gamma-2 herpesviruses.

Heterozygosity for a PAX6 deficiency (PAX6+/−) results in low levels of the PAX6 transcription factor and causes aniridia. Corneal changes in aniridia-related keratopathy (ARK) include peripheral pannus and epithelial abnormalities, which eventually result in corneal opacity and contribute to visual loss. The corneal abnormalities of Pax6+/− mice provide an excellent model for the corneal changes seen in PAX6+/− humans. The aim of the present study was to investigate the contributions of different factors (including altered cell proliferation, abnormal epithelial differentiation and incursion of the conjunctival epithelium) that may underlie the pathogenesis of the corneal changes caused by low levels of Pax6 in heterozygous Pax6+/Sey-Neu (Pax6+/−) mice. BrdU incorporation showed enhanced proliferation of Pax6+/− corneal epithelium compared to wild-type controls and analysis of p63 (a marker of high proliferative potential) revealed a slight increase in frequency of p63-positive basal corneal epithelial cells in Pax6+/− mice. Immunohistochemical investigation of K12 (a Pax6-regulated marker of corneal epithelial differentiation) in 2–52-week-old mice showed that K12 expression was delayed and down-regulated in the Pax6+/− corneal epithelium, implying that differentiation of the Pax6+/− corneal epithelium was delayed and abnormal. Goblet cells were identified within the peripheral corneal epithelium of the Pax6+/− eyes, but some were surrounded by cells expressing K12, suggesting they may have arisen in situ in the corneal epithelium. These findings suggest that low levels of Pax6 may be directly responsible for failure or delay of proper differentiation of the corneal epithelial cells, but the proliferative component of the mutant epithelium is probably not impaired. This abnormal differentiation suggests that ARK is not entirely attributable to a limbal stem cell deficiency.

An overview is provided of the cancer chemo-prevention actions of phenolic antioxidants and 6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline (ethoxyquin). These agents principally appear to exert their beneficial effects through induction of phase II drug-metabolizing enzymes such as glutathione S-transferase (GST). The requirement for oxidative metabolism of the synthetic antioxidants to carbonyl-containing compounds, including quinones, in order that they can induce gene expression is discussed. Previous work has shown that the basic leucine zipper transcription factor Nrf2 is involved in induction of GST by the phenolic antioxidant butylated hydroxyanisole (BHA). Evidence is provided from a mouse possessing a targeted disruption of the Nrf2 gene that, in murine liver, the transcription factor regulates basal expression of several class Alpha and class Mu GST subunits, but not class Pi GST. In the Nrf2 knock-out mouse, hepatic induction of class Alpha and class Mu GST by BHA and the synthetic antioxidant ethoxyquin is similarly impaired, suggesting that these agents affect gene activation by a related mechanism. Significantly, residual induction of GST by antioxidants is apparent in the Nrf2 mutant mouse, indicating the existence of an alternative mechanism of gene activation.

Kaposi’s sarcoma-associated herpesvirus (KSHV) encodes four viral interferon regulatory factors (vIRF-1–4). We investigated the mechanism and consequences of vIRF-2-mediated inhibition of interferon-response element signalling following type I interferon (IFN) induction. Western blot and electrophoretic mobility-shift assays identified the interferon-stimulated gene factor-3 (ISGF-3) components STAT1 and IRF-9 as the proximal targets of vIRF-2 activity. The biological significance of vIRF-2 inhibition of ISGF-3 was demonstrated by vIRF-2-mediated rescue of the replication of the IFN-sensitive virus encephalomyocarditis virus. This study provides both a mechanism and evidence for KSHV vIRF-2-mediated suppression of the consequences of type 1 IFN-induced signalling.

The eye develops from three major sources of cells. The surface ectoderm of the head produces the lens and the corneal, limbal, and conjunctival epithelia. The neuroectoderm produces the neural retina and retinal pigment epithelium (RPE), and extensions of both these tissues cover the ciliary body, ciliary process, and iris. The periocular mesenchyme, comprising mesoderm and neural crest–derived mesectoderm cells, produces the choroid, sclera, corneal stroma, corneal endoderm, and stroma of the ciliary body, ciliary process, and iris.1 The transcription factor Pax6 is essential for normal eye development in vertebrates.2-6 During development Pax6 is expressed in the developing lens; conjunctival, limbal and corneal epithelia; neural retina; RPE; ciliary body; and iris.4,5 After birth, Pax6 is downregulated in many eye tissues, but expression continues in the amacrine cells of the retina and the lens and in the conjunctival, limbal, and corneal epithelia.5,7,8 Homozygous Pax6−/− mice, with two nonfunctional alleles, die at birth with no eyes or nose and with brain abnormalities.5,9-11 In some cases they have abnormal dentition but the penetrance of this effect is highly dependent on the genetic background.12 Human PAX6−/− homozygotes or compound heterozygotes are rare and the condition is lethal, causing anophthalmia with severe craniofacial and central nervous system defects.13 Heterozygous Pax6+/− mice and PAX6+/− humans produce low levels of Pax6 and are viable and fertile but have a range of eye abnormalities.2 In Pax6+/− mice, abnormal eye development commonly results in small eyes, iris hypoplasia, cataracts, a thin corneal epithelium with fewer cell layers, corneal opacity, failure of the lens to separate completely from the corneal epithelium, and glaucoma. Other developmental abnormalities, including retinal dysplasia, coloboma, abnormal cell accumulation in the vitreous, and adhesions between the lens and cornea (keratolenticular strands) or between the iris and cornea (iridocorneal synechia), may also occur.10,14-18 Adult Pax6+/− mice also show progressive corneal deterioration. The corneal epithelium is thin and fragile and goblet cells accumulate; the stroma becomes vascularized from the periphery and infiltrated with inflammatory cells.16,19 Human PAX6+/− heterozygotes usually have normal-sized eyes but otherwise show a range of abnormalities similar to those in Pax6+/− mice. Clinical conditions associated with PAX6+/− heterozygosity include aniridia (absent or hypoplastic iris), Peters’ anomaly (keratolenticular adhesions with loss of posterior cornea), iridolenticular adhesions, iridocorneal adhesions, cataract, corneal opacity, congenital nystagmus, and foveal hypoplasia.2,3,13,20,21 Postnatal changes include early-onset glaucoma, corneal vascularization, corneal infiltration by inflammatory cells (autosomal dominant keratitis), and accumulation of goblet cells in the corneal epithelium. The corneal epithelium is not maintained adequately, and this is thought to involve a deficiency of limbal stem cells.22 Eye development seems to be unusually sensitive to Pax6 dosage and both PAX6 gene duplication in humans23 and experimentally induced high expression levels in the mouse24 can also cause eye abnormalities. PAX77+/− transgenic mice are hemizygous for five to six copies of a human PAX6 yeast artificial chromosome (YAC) inserted at a single locus.24 The YAC contains the human PAX6 gene with all the enhancer and promoter elements. The amino acid sequence of human PAX6 is identical with mouse Pax6 and the human PAX6 regulatory elements are fully functional in mouse, so that the transgene is expressed in an appropriate tissue-specific manner and fully functional.24 The extra copies of PAX6 can compensate for the Pax6 deficiencies in both Pax6−/− and Pax6+/− mice. On a wild-type Pax6+/+ genetic background, however, this overexpression of Pax6 in PAX77+/− mice results in eye abnormalities, which overlap with those seen in Pax6+/− mice. These hemizygous PAX77+/− Pax6+/+ mice often had microphthalmia, small corneas; flat irides; abnormal, small or absent ciliary bodies; small abnormal lenses; and abnormal photoreceptors, but the phenotype was variable. Pax6 has multiple pleiotropic roles in cell proliferation, migration, adhesion, and signaling25 and may act cell-autonomously or nonautonomously. Experimental studies have identified several primary cell-autonomous roles for Pax6 that underlie the eye abnormalities in homozygous Pax6−/− mice.6,26-30 Some of the eye abnormalities in heterozygous Pax6+/− mice are caused by nonautonomous effects and may be secondary to primary affects of low Pax6 in the surface ectoderm and lens.14,15 Nonautonomous effects probably also underlie some of the defects caused by abnormal migration of neural crest cells18 but cell-autonomous effects may also be involved because Pax6 appears to be expressed transiently in the corneal stroma,17 and chimera experiments indicate that Pax6 acts cell-autonomously in this tissue.28 Analysis of mouse chimeras has been used successfully to distinguish between the cell-autonomous and nonautonomous effects of Pax6−/− and Pax6+/− genotypes on eye development.14,26-28 One important observation to emerge from studies of Pax6+/−↔wild-type chimeras was that Pax6+/− cells were excluded from the lens epithelium of Pax6+/−↔wild-type chimeras by embryonic day (E)16.5. Although Pax6+/− cells contributed to tissues of the anterior segment, the chimeras did not show iris hypoplasia or other anterior segment abnormalities that occur in nonchimeric Pax6+/− mice. This exclusion of Pax6+/− cells from the lens epithelium is interpreted as a cell-autonomous effect, and it produces an almost entirely wild-type lens. It was further proposed that the largely wild-type lens cells exerted a nonautonomous effect on the anterior segment and prevented the formation of anterior segment abnormalities.14 One of the purposes of this study was to determine whether PAX77+/− cells are similarly excluded from the lens. In the present study, we first examined the range of eye abnormalities caused by the PAX77+/− genotype on different genetic backgrounds. We then analyzed fetal PAX77+/−↔wild-type chimeras to investigate the underlying developmental causes of these eye abnormalities and distinguish between cell-autonomous and nonautonomous effects of elevated Pax6 expression in the eye. The use of these chimeras to demonstrate a cell-autonomous effect of increased Pax6 expression in the neurocortex has been reported elsewhere.31 Both low levels of Pax6 in Pax6+/− heterozygotes and high levels in PAX77+/− transgenics cause lens abnormalities, and so we specifically wanted to use chimeras to test the hypothesis that PAX77+/− cells would be depleted or excluded from the lens epithelium, like Pax6+/− cells in our Pax6+/−↔wild-type chimeras.

The diversity of viral strategies to modulate complement activation indicates that this component of the immune system has significant antiviral potential. One example is the Kaposi's sarcoma-associated herpesvirus (KSHV) complement control protein (KCP), which inhibits progression of the complement cascade. Rhesus rhadinovirus (RRV), like KSHV, is a member of the subfamilyGammaherpesvirinaeand currently provides the only in vivo model of KSHV pathobiology in primates. In the present study, we characterized the KCP homologue encoded by RRV, RRV complement control protein (RCP). Two strains of RRV have been sequenced to date (H26-95 and 17577), and the RCPs they encode differ substantially in structure: RCP from strain H26-95 has four complement control protein (CCP) domains, whereas RCP from strain 17577 has eight CCP domains. Transcriptional analyses of the RCP gene (ORF4, referred to herein asRCP) in infected rhesus macaque fibroblasts mapped the ends of the transcripts of both strains. They revealed that H26-95 encodes a full-length, unsplicedRCPtranscript, while 17577RCPgenerates a full-length unspliced mRNA and two alternatively spliced transcripts. Western blotting confirmed that infected cells express RCP, and immune electron microscopy disclosed this protein on the surface of RRV virions. Functional studies of RCP encoded by both RRV strains revealed their ability to suppress complement activation by the classical (antibody-mediated) pathway. These data provide the foundation for studies into the biological significance of gammaherpesvirus complement regulatory proteins in a tractable, non-human primate model.

Levels of expression of the transcription factor Pax6 vary throughout corticogenesis in a rostro-lateralhigh to caudo-mediallow gradient across the cortical proliferative zone. Previous loss-of-function studies have indicated that Pax6 is required for normal cortical progenitor proliferation, neuronal differentiation, cortical lamination and cortical arealization, but whether and how its level of expression affects its function is unclear. We studied the developing cortex of PAX77 YAC transgenic mice carrying several copies of the human PAX6 locus with its full complement of regulatory regions. We found that PAX77 embryos express Pax6 in a normal spatial pattern, with levels up to three times higher than wild type. By crossing PAX77 mice with a new YAC transgenic line that reports Pax6 expression (DTy54), we showed that increased expression is limited by negative autoregulation. Increased expression reduces proliferation of late cortical progenitors specifically, and analysis of PAX77↔wild-type chimeras indicates that the defect is cell autonomous. We analyzed cortical arealization in PAX77 mice and found that, whereas the loss of Pax6 shifts caudal cortical areas rostrally, Pax6 overexpression at levels predicted to shift rostral areas caudally has very little effect. These findings indicate that Pax6 levels are stabilized by autoregulation, that the proliferation of cortical progenitors is sensitive to altered Pax6 levels and that cortical arealization is not.

Purpose To investigate the etiology of corneal dysfunction in the Pax6(+/-) mouse model of aniridia-related keratopathy. Methods Mosaic patterns of X-gal staining were compared in the corneal and limbal epithelia of female Pax6(+/-) and Pax6(+/+) littermates, age 3 to 28 weeks, hemizygous for an X-linked LacZ transgene, and Pax6(+/+), LacZ(-) Pax6(+/+), LacZ(+) and Pax6(+/+), LacZ(-) Pax6(+/-), LacZ(+) chimeras. Histologic examination of chimeric corneas was performed. Results Disrupted patterns of X-gal staining showed that heterozygosity for Pax6 perturbed clonal patterns of growth and development in the corneal and limbal epithelium. Centripetal migration of Pax6(+/-) corneal epithelial cells was diverted. Normal patterns of centripetal Pax6(+/-) cell migration and epithelial morphology were restored in Pax6(+/+) Pax6(+/-) chimeras. Fewer, larger clones of limbal stem cells were present in Pax6(+/-) eyes, compared with wild-type. In the chimeras, Pax6(+/-) limbal stem cells were cell-autonomously depleted or less efficient than wild-type cells at producing progeny to populate the corneal epithelium. Conclusions The correct Pax6 dosage is necessary for normal clonal growth during corneal development, normal limbal stem cell activity, and correct corneal epithelial cell migration. Disruption of normal cell movement in heterozygotes may be the consequence of failure of nonautonomous guidance cues. Degeneration of the corneal surface in aniridia-related keratopathy relates to both a deficiency within the limbal stem cell niche and nonautonomous diversion of corneal epithelial cell migration.

Mice that lack the Nrf2 basic-region leucine-zipper transcription factor are more sensitive than wild-type (WT) animals to the cytotoxic and genotoxic effects of foreign chemicals and oxidants. To determine the basis for the decrease in tolerance of the Nrf2 homozygous null mice to xenobiotics, enzyme assay, Western blotting and gene-specific real-time PCR (TaqMan®) have been used to examine the extent to which hepatic expression of GSH-dependent enzymes is influenced by the transcription factor. The amounts of protein and mRNA for class Alpha, Mu and Pi glutathione S-transferases were compared between WT and Nrf2 knockout (KO) mice of both sexes under both constitutive and inducible conditions. Among the class Alpha and class Mu transferases, constitutive expression of Gsta1, Gsta2, Gstm1, Gstm2, Gstm3, Gstm4 and Gstm6 subunits was reduced in the livers of Nrf2 mutant mice to between 3% and 60% of that observed in WT mice. Induction of these subunits by butylated hydroxyanisole (BHA) was more marked in WT female mice than in WT male mice. TaqMan® analyses showed the increase in transferase mRNA caused by BHA was attenuated in Nrf2−/− mice, with the effect being most apparent in the case of Gsta1, Gstm1 and Gstm3. Amongst class Pi transferase subunits, the constitutive hepatic level of mRNA for Gstp1 and Gstp2 was not substantially affected in the KO mice, but their induction by BHA was dependent on Nrf2; this was more obvious in female mutant mice than in male mice. Nrf2 KO mice exhibited reduced constitutive expression of the glutamate cysteine ligase catalytic subunit, and, to a lesser extent, the expression of glutamate cysteine ligase modifier subunit. Little variation was observed in the levels of glutathione synthase in the different mouse lines. Thus the increased sensitivity of Nrf2−/− mice to xenobiotics can be partly attributed to a loss in constitutive expression of multiple GSH-dependent enzymes, which causes a reduction in intrinsic detoxification capacity in the KO animal. These data also indicate that attenuated induction of GSH-dependent enzymes in Nrf2−/− mice probably accounts for their failure to adapt to chronic exposure to chemical and oxidative stress.