Stability of murine cytomegalovirus genome after in vitro and in vivo passage.

While large DNA viruses are thought to have low mutation rates, only a small fraction of their genomes have been analyzed at the single-nucleotide level. Here, we defined the genetic stability of murine cytomegalovirus (MCMV) by whole-genome sequencing. Independently assembled sequences of three sister plaques showed only two single-base-pair substitutions after in vitro passage. In vivo-passaged MCMV likewise demonstrated low mutation rates, comparable to those after in vitro passage, indicating high genome stability of MCMV at the single-nucleotide level in the absence of obvious selection pressure.

Evidence for high bi-allelic expression of activating Ly49 receptors.

Stochastic expression is a hallmark of the Ly49 family that encode the main MHC class-I-recognizing receptors of mouse natural killer (NK) cells. This highly polygenic and polymorphic family includes both activating and inhibitory receptor genes and is one of genome's fastest evolving loci. The inhibitory Ly49 genes are expressed in a stochastic mono-allelic manner, possibly under the control of an upstream bi-directional early promoter and show mono-allelic DNA methylation patterns. To date, no studies have directly addressed the transcriptional regulation of the activating Ly49 receptors. Our study shows differences in DNA methylation pattern between activating and inhibitory genes in C57BL/6 and F1 hybrid mouse strains. We also show a bias towards bi-allelic expression of the activating receptors based on allele-specific single-cell RT-PCR in F1 hybrid NK cells for Ly49d and Ly49H expression in Ly49h(+/-) mice. Furthermore, we have identified a region of high sequence identity with possible transcriptional regulatory capacity for the activating Ly49 genes. Our results also point to a likely difference between NK and T-cells in their ability to transcribe the activating Ly49 genes. These studies highlight the complex regulation of this rapidly evolving gene family of central importance in mouse NK cell function.

Immunologic rheumatic disorders.

We provide the basics for clinicians who might be called on to consider the diagnosis of diseases such as systemic lupus erythematosus (SLE) or rheumatoid arthritis (RA) in their practice. We will emphasize clinical recognition and first-line laboratory testing. Only characteristics of the classic rheumatic inflammatory diseases (ie, RA, seronegative spondyloarthropathy, SLE, antiphospholipid syndrome, Sjögren syndrome, scleroderma, and polymyositis/dermatomyositis) will be covered. In the past decade, treatment for RA and seronegative spondyloarthropathy has substantially improved. Their treatment has been revolutionized by the use of methotrexate and, more recently, TNF inhibitors, T-cell costimulation modulators, and B-cell depletion. The goal of RA treatment today is to induce a complete remission as early as possible in the disease process, with the mantra being "elimination of synovitis equals elimination of joint destruction." The hope is that if the major mediators of Sjögren syndrome, SLE, or scleroderma can be identified and then blocked, as in the example of TNF inhibitors in patients with RA, more specific treatments will become available. Thus RA has become an excellent model of this evolving paradigm. Through the identification of major mediators in its pathogenesis, novel and highly efficacious therapeutic agents have been developed.

Cytohesin binder and regulator (cybr) is not essential for T- and dendritic-cell activation and differentiation.

Cybr (also known as Cytip, CASP, and PSCDBP) is an interleukin-12-induced gene expressed exclusively in hematopoietic cells and tissues that associates with Arf guanine nucleotide exchange factors known as cytohesins. Cybr levels are dynamically regulated during T-cell development in the thymus and upon activation of peripheral T cells. In addition, Cybr is induced in activated dendritic cells and has been reported to regulate dendritic cell (DC)-T-cell adhesion. Here we report the generation and characterization of Cybr-deficient mice. Despite the selective expression in hematopoietic cells, there was no intrinsic defect in T- or B-cell development or function in Cybr-deficient mice. The adoptive transfer of Cybr-deficient DCs showed that they migrated efficiently and stimulated proliferation and cytokine production by T cells in vivo. However, competitive stem cell repopulation experiments showed a defect in the abilities of Cybr-deficient T cells to develop in the presence of wild-type precursors. These data suggest that Cybr is not absolutely required for hematopoietic cell development or function, but stem cells lacking Cybr are at a developmental disadvantage compared to wild-type cells. Collectively, these data demonstrate that despite its selective expression in hematopoietic cells, the role of Cybr is limited or largely redundant. Previous in vitro studies using overexpression or short interfering RNA inhibition of the levels of Cybr protein appear to have overestimated its immunological role.

Ly49h is necessary for genetic resistance to murine cytomegalovirus.

Natural killer (NK) cells play critical roles in antiviral immunity. While the importance of effector mechanisms such as interferons has been demonstrated through knockout mice, specific mechanisms of how viruses are recognized and controlled by NK cells are less well defined. Previous genetic studies have mapped the resistance genes for murine cytomegalovirus (MCMV), herpes simplex virus-1 (HSV-1), and ectromelia virus to the NK gene complex on murine chromosome 6, a region containing the polymorphic Ly49 and Nkrp1 families. Genetic resistance to MCMV in C57BL/6 has been attributed to Ly49H, an activation receptor, through susceptibility of the recombinant inbred strain BXD-8 that lacks Ly49h (also known as Klra8) but derived about half of its genome from its DBA/2 progenitor. However, it remained possible that epigenetic effects could account for the MCMV phenotype in BXD-8 mice. Herein, we report the generation of a novel congenic murine strain, B6.BXD8-Klra8 ( Cmv1-del )/Wum, on the C57BL/6 genetic background to evaluate the effect of deletion of a single NK activation receptor, Ly49H. Deletion of Ly49H rendered mice much more susceptible to MCMV infection. This increase in susceptibility did not appear to be a result of a difference in NK cell expansion or interferon-gamma (IFN-gamma) production between the C57BL/6 and the B6.BXD8 strains. On the other hand, the deletion of Ly49h did not otherwise affect NK cell maturation or Ly49D expression and had no effect on susceptibility to HSV-1 or ectromelia virus. In conclusion, Ly49h is necessary for genetic resistance to MCMV, but not HSV-1 or ectromelia virus.

Continuous engagement of a self-specific activation receptor induces NK cell tolerance.

Natural killer (NK) cell tolerance mechanisms are incompletely understood. One possibility is that they possess self-specific activation receptors that result in hyporesponsiveness unless modulated by self-major histocompatability complex (MHC)-specific inhibitory receptors. As putative self-specific activation receptors have not been well characterized, we studied a transgenic C57BL/6 mouse that ubiquitously expresses m157 (m157-Tg), which is the murine cytomegalovirus (MCMV)-encoded ligand for the Ly49H NK cell activation receptor. The transgenic mice were more susceptible to MCMV infection and were unable to reject m157-Tg bone marrow, suggesting defects in Ly49H(+) NK cells. There was a reversible hyporesponsiveness of Ly49H(+) NK cells that extended to Ly49H-independent stimuli. Continuous Ly49H-m157 interaction was necessary for the functional defects. Interestingly, functional defects occurred when mature wild-type NK cells were adoptively transferred to m157-Tg mice, suggesting that mature NK cells may acquire hyporesponsiveness. Importantly, NK cell tolerance caused by Ly49H-m157 interaction was similar in NK cells regardless of expression of Ly49C, an inhibitory receptor specific for a self-MHC allele in C57BL/6 mice. Thus, engagement of self-specific activation receptors in vivo induces an NK cell tolerance effect that is not affected by self-MHC-specific inhibitory receptors.

Vanadate, an inhibitor of stromelysin and collagenase expression, suppresses collagen induced arthritis.

OBJECTIVE: Collagen induced arthritis (CIA) is a model of chronic inflammatory synovitis with pannus, neovascularization, and joint destruction similar to rheumatoid arthritis (RA). Matrix metalloproteinases (MMP) are involved in degradation of the extracellular matrix and joint destruction in RA. c-fos and c-jun are protooncogenes whose products combine to form activating protein (AP-1), a regulatory protein that is required for cell proliferation and the transcription of a variety of genes, including MMP such as collagenase and stromelysin. Administration of vanadium compounds suppresses c-fos/c-jun expression and AP-1 activity, resulting in inhibition of MMP expression in response to factors such as interleukin 1 (IL-1). We evaluated whether a vanadium AP-1 inhibitor could reduce MMP expression and subsequent joint damage in CIA. METHODS: Vanadate [bis (maltolato) oxovanadium (IV) (BMOV; 10 mg/kg/day)] and the reducing agent N-acetyl cysteine (NAC; 100 mg/kg/day) were given subcutaneously daily in an attempt to suppress established CIA in rats. NAC in combination with vanadate appeared to increase the efficacy of c-fos/c-jun inhibition, while decreasing toxicity. Controls were given NAC alone. Clinical, radiographic, and histologic measures were evaluated as well as synovial MMP and IL-1a expression. RESULTS: BMOV therapy, initiated on the day of onset of clinical arthritis, significantly reduced clinical arthritis within 2 days (p <0.05) compared to controls. Significance was maintained to the termination of the study on Day 18 post-arthritis onset (p < 0.005), with a maximum difference seen on Day 5 (p < 0.00001). Blinded radiographic scores at the completion of the protocols indicated less joint destruction in the experimental group compared to the control group (p < 0.005). Scanning and transmission electron microscopy confirmed the preservation of articular cartilage with therapy. In BMOV-treated rats, synovial mRNA expression of collagenase, stromelysin, and IL-la were reduced by 78%, 58%, and 85%, respectively, compared to controls. CONCLUSION: This is the first study of vanadate as a potential antirheumatic agent. Further study of this AP-1 and MMP inhibitor may lead to new treatment options in RA.

Constitutive activation of the neuregulin-1/erbB signaling pathway promotes the proliferation of a human peripheral neuroepithelioma cell line.

Neuregulin-1 (NRG-1) proteins, acting through their erbB receptors, promote the differentiation, survival and/or proliferation of many cell types in the developing nervous system, including neural crest cells and neural crest-derived Schwann cells. We have recently found that the proliferation of a neoplastic Schwann cell line is dependent on constitutive activation of the NRG-1/erbB signaling pathway and that overexpression of NRG-1 in myelinating Schwann cells induces the formation of malignant peripheral nerve sheath tumors. These observations suggested that NRG-1 might similarly promote mitogenesis in a variety of neural neoplasms including peripheral neuroepitheliomas, aggressive neural crest-derived neoplasms that arise in nerves and soft tissues. To test this hypothesis, we examined the expression of NRG-1 and its erbB receptors in SK-N-MC neuroepithelioma cells. SK-N-MC cells expressed multiple NRG-1 proteins and mRNAs encoding several alpha and beta isoforms from the sensory and motor neuron-derived factor NRG-1 subfamily as well as the NRG-1 receptor subunits erbB2, erbB3, and erbB4. The erbB receptors expressed by SK-N-MC cells were constitutively tyrosine phosphorylated and inhibiting these kinases with the erbB specific inhibitor PD158780 reduced SK-N-MC DNA synthesis in a dose-dependent manner. We conclude that constitutive activation of the NRG-1/erbB signaling pathway promotes the proliferation of SK-N-MC neuroepithelioma cells in vitro and hypothesize that NRG-1/erbB autocrine, paracrine or juxtacrine signaling may contribute to the development and/or progression of neuroepitheliomas in vivo.

Cybr, a cytokine-inducible protein that binds cytohesin-1 and regulates its activity.

Cytokines regulate lymphocyte development and differentiation, but precisely how they control these processes is still poorly understood. By using microarray technology to detect cytokine-induced genes, we identified a cDNA encoding Cybr, which was increased markedly in cells incubated with IL-2 and IL-12. The mRNA was most abundant in hematopoietic cells and tissues. The predicted amino acid sequence is similar to that of GRP-1-associated protein (GRASP), a recently identified retinoic acid-induced cytohesin-binding protein. Physical interaction, dependent on the coiled-coil domains of Cybr and cytohesin-1, was demonstrated by coimmunoprecipitation of the overexpressed proteins from 293T cells. Cytohesin-1, in addition to its role in cell adhesion, is a guanine nucleotide-exchange protein activator of ARF GTPases. Acceleration of guanosine 5prime prime or minute-O-(thiotriphosphate) binding to ARF by cytohesin-1 in vitro was enhanced by Cybr. Because the binding protein modified activation of ADP ribosylation factor by cytohesin-1, we designate this cytokine-inducible protein Cybr (cytohesin binder and regulator).