The association of PTPN22 rs2476601 with juvenile idiopathic arthritis is specific to females.

A preponderance of females develop autoimmune disease, including juvenile idiopathic arthritis (JIA), yet the reason for this bias remains elusive. Evidence suggests that genetic risk of disease may be influenced by sex. PTPN22 rs2476601 is associated with JIA and numerous other autoimmune diseases, and has been reported to show female-specific association with type 1 diabetes. We performed main effect and sex-stratified association analyses to determine whether a sex-specific association exists in JIA. As expected, rs2476601 was associated with JIA in our discovery (413 cases and 690 controls) and replication (1008 cases and 9284 controls) samples. Discovery sample sex-stratified analyses demonstrated an association specifically in females (odds ratio (OR)=2.35, 95% confidence interval (CI)=1.52-3.63, P=0.00011) but not males (OR=0.91, 95% CI=0.52-1.60, P=0.75). This was similarly observed in the replication sample. There was evidence for genotype-by-sex interaction (Pinteraction=0.009). The association between rs2476601 and JIA appears restricted to females, partly accounting for the predominance of females with this disease.

Oxygen radicals and signaling.

Recent evidence suggests that reactive oxygen species, such as superoxide anions and hydrogen peroxide, function as intracellular second messengers. This review will discuss the progress in understanding the intracellular pathways leading from ligand stimulation to the generation of oxidants, as well as some of the increasing number of cellular processes that appear to be subject to redox regulation.

Apoptosis occurs predominantly in bystander cells and not in productively infected cells of HIV- and SIV-infected lymph nodes.

Although 13 years have passed since identification of human immunodeficiency virus-1 (HIV-1) as the cause of AIDS, we do not yet know how HIV kills its primary target, the T cell that carries the CD4 antigen. We and others have shown an increase in the percentage of apoptotic cells among circulating CD4+ (and CD8+) T cells of HIV-seropositive individuals and an increase in frequency of apoptosis with disease progression. However, it is not known if this apoptosis occurs in infected or uninfected T cells. We show here, using in situ labelling of lymph nodes from HIV-infected children and SIV-infected macaques, that apoptosis occurs predominantly in bystander cells and not in the productively infected cells themselves. These data have implications for pathogenesis and therapy, namely, arguing that rational drug therapy may involve combination agents targeting viral replication in infected cells and apoptosis of uninfected cells.

Surface expression of a T cell receptor beta (TCR-beta) chain in the absence of TCR-alpha, -delta, and -gamma proteins.

The antigen receptor expressed by mature T cells has been described as a disulfide-linked alpha/beta or gamma/delta heterodimer noncovalently associated with CD3, a complex of transmembrane proteins that communicates signals from the T cell receptor (TCR) to the cell interior. Studies suggest that all component chains must assemble intracellularly before surface expression can be achieved. We described, however, a CD4+/CD8+ transformed murine thymocyte, KKF, that expresses surface TCR-beta chains in the absence of gamma, delta, and alpha proteins; these beta chains are only weakly associated with CD3-epsilon and CD3-zeta. Furthermore, KKF responds differently to stimulation through TCR-beta and CD3-epsilon, a functional dissociation that has been ascribed to a CD4+/CD8+ subpopulation of normal thymocytes. KKF's unique TCR structure may offer an explanation for the functional anomalies observed.

Crosslinking CD4 by human immunodeficiency virus gp120 primes T cells for activation-induced apoptosis.

During human immunodeficiency virus (HIV) infection there is a profound and selective decrease in the CD4+ population of T lymphocytes. The mechanism of this depletion is not understood, as only a small fraction of all CD4+ cells appear to be productively infected with HIV-1 in seropositive individuals. In the present study, crosslinking of bound gp120 on human CD4+ T cells followed by signaling through the T cell receptor for antigen was found to result in activation-dependent cell death by a form of cell suicide termed apoptosis, or programmed cell death. The data indicate that even picomolar concentrations of gp120 prime T cells for activation-induced cell death, suggesting a mechanism for CD4+ T cell depletion in acquired immune deficiency syndrome (AIDS), particularly in the face of concurrent infection and antigenic challenge with other organisms. These results also provide an explanation for the enhancement of infection by certain antibodies against HIV, and for the paradox that HIV appears to cause AIDS after the onset of antiviral immunity.

Requirement for generation of H2O2 for platelet-derived growth factor signal transduction.

Stimulation of rat vascular smooth muscle cells (VSMCs) by platelet-derived growth factor (PDGF) transiently increased the intracellular concentration of hydrogen peroxide (H2O2). This increase could be blunted by increasing the intracellular concentration of the scavenging enzyme catalase or by the chemical antioxidant N-acetylcysteine. The response of VSMCs to PDGF, which includes tyrosine phosphorylation, mitogen-activated protein kinase stimulation, DNA synthesis, and chemotaxis, was inhibited when the growth factor-stimulated rise in H2O2 concentration was blocked. These results suggest that H2O2 may act as a signal-transducing molecule, and they suggest a potential mechanism for the cardioprotective effects of antioxidants.

Potential role of human cytomegalovirus and p53 interaction in coronary restenosis.

A subset of patients who have undergone coronary angioplasty develop restenosis, a vessel renarrowing characterized by excessive proliferation of smooth muscle cells (SMCs). Of 60 human restenosis lesions examined, 23 (38 percent) were found to have accumulated high amounts of the tumor suppressor protein p53, and this correlated with the presence of human cytomegalovirus (HCMV) in the lesions. SMCs grown from the lesions expressed HCMV protein IE84 and high amounts of p53. HCMV infection of cultured SMCs enhanced p53 accumulation, which correlated temporally with IE84 expression. IE84 also bound to p53 and abolished its ability to transcriptionally activate a reporter gene. Thus, HCMV, and IE84-mediated inhibition of p53 function, may contribute to the development of restenosis.

Mitogenic signaling mediated by oxidants in Ras-transformed fibroblasts.

NIH 3T3 fibroblasts stably transformed with a constitutively active isoform of p21(Ras), H-RasV12 (v-H-Ras or EJ-Ras), produced large amounts of the reactive oxygen species superoxide (.O2-). .O2- production was suppressed by the expression of dominant negative isoforms of Ras or Rac1, as well as by treatment with a farnesyltransferase inhibitor or with diphenylene iodonium, a flavoprotein inhibitor. The mitogenic activity of cells expressing H-RasV12 was inhibited by treatment with the chemical antioxidant N-acetyl-L-cysteine. Mitogen-activated protein kinase (MAPK) activity was decreased and c-Jun N-terminal kinase (JNK) was not activated in H-RasV12-transformed cells. Thus, H-RasV12-induced transformation can lead to the production of .O2- through one or more pathways involving a flavoprotein and Rac1. The implication of a reactive oxygen species, probably .O2-, as a mediator of Ras-induced cell cycle progression independent of MAPK and JNK suggests a possible mechanism for the effects of antioxidants against Ras-induced cellular transformation.

Regulation of endothelial cell adhesion by profilin.

BACKGROUND: Although profilin is believed to be an essential regulator of the actin cytoskeleton in most cells, its precise role in mammalian cells remains unknown. We have used replication-incompetent adenovirus carrying the human profilin I cDNA as a means rapidly to increase the concentration of profilin in human aortic endothelial cells 12-31-fold above baseline--levels never before achieved in mammalian cells. RESULTS: The concentration of filamentous actin was not detectably affected by profilin overexpression. Actin stress fibers were, however, absent from areas of high profilin content in overexpressing cells, and the bulk of filaments was located at the periphery of the cells. We observed a gradient in the distribution of overexpressed profilin in migrating endothelial cells, with most profilin molecules concentrated near the advancing edge where focal contacts are being formed and focal adhesion proteins are located. Profilin overexpression resulted in increased recruitment of fibronectin receptors to the plasma membrane. Adhesion of endothelial cells to fibronectin was markedly and selectively increased by profilin overexpression. CONCLUSIONS: We conclude that an important role for profilin in mammalian cells may be its contribution to the formation of focal contacts, particularly those involving the fibronectin receptor.

Human cytomegalovirus increases modified low density lipoprotein uptake and scavenger receptor mRNA expression in vascular smooth muscle cells.

Evidence suggests a possible role for human cytomegalovirus (HCMV) in the development of arteriosclerosis. One of the earliest events in plaque formation is the accumulation of lipid-laden foam cells, derived from macrophages and smooth muscle cells (SMCs). The lipid accumulation that occurs depends upon the uptake of oxidized LDL (Ox-LDL), a process in which the scavenger receptor (SR) has been postulated to play an important role. We therefore examined the effects of HCMV on this process. We demonstrate that HCMV infection of human SMCs increases modified LDL uptake and stimulates class A SR gene (SR-A) mRNA expression. In addition, infection of rat SMCs with HCMV, which causes immediate early gene expression (IE72/IE84), but no early or late HCMV gene products and no cytopathic effects, also increases SMC uptake of Ox-LDL and acetylated LDL, with either effect blocked by an excess of either cold Ox-LDL or acetylated-LDL, and by fucoidin, an SR competitor. Cotransfection of an IE72, but not an IE84, expression plasmid and a plasmid containing a Class A SR promoter/reporter gene construct enhances SR promoter activity. Since increased Ox-LDL uptake is believed to play an important role in arteriosclerosis, these results provide a link between HCMV infection and arteriosclerotic plaque formation.

A requirement for the rac1 GTPase in the signal transduction pathway leading to cardiac myocyte hypertrophy.

We have used adenoviral-mediated gene transfer of a constitutively active (V12rac1) and dominant negative (N17rac1) isoform of rac1 to assess the role of this small GTPase in cardiac myocyte hypertrophy. Expression of V12rac1 in neonatal cardiac myocytes results in sarcomeric reorganization and an increase in cell size that is indistinguishable from ligand-stimulated hypertrophy. In addition, V12rac1 expression leads to an increase in atrial natriuretic peptide secretion. In contrast, expression of N17rac1, but not a truncated form of Raf-1, attenuated the morphological hypertrophy associated with phenylephrine stimulation. Consistent with the observed effects on morphology, expression of V12rac1 resulted in an increase in new protein synthesis, while N17rac1 expression inhibited phenylephrine-induced leucine incorporation. These results suggest rac1 is an essential element of the signaling pathway leading to cardiac myocyte hypertrophy.

Protection from reoxygenation injury by inhibition of rac1.

We demonstrate that adenoviral-mediated gene transfer of a dominant negative rac1 gene product (N17rac1) inhibits the intracellular burst of reactive oxygen species (ROS) that occurs after reoxygenation of vascular smooth muscle cells. In contrast, expression of a dominant negative ras gene (N17ras) had no effect. Challenge of control cells and cells expressing N17rac1 with a direct oxidant stress produced an equivalent increase in intracellular ROS levels and subsequent cell death. This suggests that N17rac1 expression appears to block production of harmful oxygen radicals and does not act directly or indirectly to scavenge ROS generated during reoxygenation. Expression of N17rac1 results in protection from hypoxia/reoxygenation-induced cell death in a variety of cell types including vascular smooth muscle cells, fibroblasts, endothelial cells, and ventricular myocytes. These results suggest that reoxygenation injury requires the activation of rac proteins, and that inhibition of rac-dependent pathways may be a useful strategy for the prevention of reperfusion injury in ischemic tissues.

Indirect mechanisms of HIV pathogenesis: how does HIV kill T cells?

Although twelve years have passed since the identification of HIV as the cause of AIDS, we do not yet know how HIV kills its target, the CD4+ T cell, nor how this killing cripples the immune system. Prominent theories include direct killing of infected CD4+ T cells by the action or accumulation of cytopathic viral DNA, transcripts or proteins, or by virus-specific cytotoxic T lymphocytes, and indirect killing of uninfected CD4+ T cells (and other immune cells) by autoimmune mechanisms, cytokines, superantigens, or apoptosis. In the past year, studies have provided tantalizing clues as to why infected cells may not die and how these infected cells kill innocent bystander cells.

Role for mitochondrial oxidants as regulators of cellular metabolism.

Leakage of mitochondrial oxidants contributes to a variety of harmful conditions ranging from neurodegenerative diseases to cellular senescence. We describe here, however, a physiological and heretofore unrecognized role for mitochondrial oxidant release. Mitochondrial metabolism of pyruvate is demonstrated to activate the c-Jun N-terminal kinase (JNK). This metabolite-induced rise in cytosolic JNK1 activity is shown to be triggered by increased release of mitochondrial H(2)O(2). We further demonstrate that in turn, the redox-dependent activation of JNK1 feeds back and inhibits the activity of the metabolic enzymes glycogen synthase kinase 3beta and glycogen synthase. As such, these results demonstrate a novel metabolic regulatory pathway activated by mitochondrial oxidants. In addition, they suggest that although chronic oxidant production may have deleterious effects, mitochondrial oxidants can also function acutely as signaling molecules to provide communication between the mitochondria and the cytosol.

rac1 regulates a cytokine-stimulated, redox-dependent pathway necessary for NF-kappaB activation.

The signal transduction pathway leading to the activation of the transcription factor NF-kappaB remains incompletely characterized. We demonstrate that in HeLa cells, transient expression of a constitutively active mutant of the small GTP-binding protein rac1 (V12rac1) leads to a significant increase in NF-kappaB transcriptional activity. In addition, expression of a dominant-negative rac1 mutant (N17rac1) inhibits basal and interleukin 1beta-stimulated NF-kappaB activity. Gel shift analysis using nuclear extract prepared from HeLa cells infected with a recombinant adenovirus encoding N17rac1 (Ad.N17racl) showed reduced levels of cytokine-stimulated DNA binding to a consensus NF-kappaB binding site. We demonstrate that rac proteins function downstream of ras proteins in the activation of NF-kappaB. In addition, V12rac1 stimulation of NF-kappaB activity is shown to be independent of the ability of rac proteins to activate the family of c-jun amino-terminal kinases. In an effort to further explore how rac proteins might regulate NF-kappaB activity, we demonstrate that expression of V12rac1 in HeLa cells or stimulation with cytokine results in a significant increase in intracellular reactive oxygen species (ROS). Treatment of cells with either of two chemically unrelated antioxidants inhibits the rise in ROS that occurs following V12rac1 expression as well as the ability of V12rac1 to stimulate NF-kappaB activity. These results suggest that in HeLa cells, rac1 regulates intracellular ROS production and that rac proteins function as part of a redox-dependent signal transduction pathway leading to NF-kappaB activation.

Endothelial progenitor cells as putative targets for angiostatin.

Angiostatin, a product of the proteolytic cleavage of plasminogen, possesses potent antitumor and antiangiogenic properties in vivo. Studies with cultured endothelial cells suggest that under certain conditions, angiostatin inhibits the migration and proliferation of these cells or, alternatively, increases their rate of apoptosis. In general, the effects of angiostatin have been considerably less potent in vitro than in vivo. One potential explanation for this disparity is that the in vivo target of angiostatin is not the mature endothelial cell. Recently, evidence has accumulated to show that circulating endothelial progenitor cells (EPCs) contribute to neovascularization. In this study, we have isolated EPCs from human subjects and demonstrated that, in contrast to that of mature endothelial cells, the growth of EPCs is exquisitely sensitive to angiostatin. These results suggest that angiostatin and related compounds may exert their biological effects by inhibiting the contribution of EPCs to angiogenesis and not by altering the growth of mature endothelial cells.

Vascular effects following homozygous disruption of p47(phox) : An essential component of NADPH oxidase.

BACKGROUND: Evidence suggests that the vessel wall contains an oxidase similar, if not identical, to phagocytic NADPH oxidase. We tested the contribution of this specific oxidase to the progression of atherosclerosis and the regulation of blood pressure. METHODS AND RESULTS: An examination of aortic rings from wild-type mice and mice with homozygous targeted disruptions in p47(phox) revealed that p47(phox) knockout mice had a reduction in vascular superoxide production. However, analyses of apoE -/- p47(phox)+/+ and apoE -/- p47(phox) -/- strains of mice demonstrated no significant differences in atherosclerotic lesion sizes. Similarly, analyses of wild-type and p47(phox) knockout mice revealed no differences in either basal blood pressure or the rise in blood pressure seen after the pharmacological inhibition of nitric oxide synthase. CONCLUSIONS: NADPH oxidase contributes to basal vascular superoxide production. However, the absence of a functional oxidase does not significantly affect the progression of atherosclerosis in the standard mouse apoE -/- model, nor does it significantly influence basal blood pressure.

Cytomegalovirus infection of rats increases the neointimal response to vascular injury without consistent evidence of direct infection of the vascular wall.

BACKGROUND: Previous studies suggest that infection may play a role in restenosis and atherogenesis; cytomegalovirus (CMV) is one of the implicated pathogens. To determine a potential causal role of CMV in these disease processes, we assessed whether CMV infection increases the neointimal response to injury of the rat carotid artery. METHODS AND RESULTS: Carotid injury was performed on 60 rats; immediately thereafter, 30 rats were infected with rat CMV, and the other 30 were mock-infected. Six weeks later, rats were euthanized, and the salivary glands, spleen, and carotid arteries were harvested. CMV infection was associated with significant exacerbation of the neointimal response to injury (neointimal to medial ratio 0.81+/-0. 59 versus 0.31+/-0.38 in CMV-infected versus control rats; P<0.0001). This occurred despite absence of infectious virus from vascular tissues and detection of CMV DNA by polymerase chain reaction in the injured artery only at day 3 after infection. Persistent distant infection, associated with systemic cytokine response, was evidenced by isolation of infectious virus from homogenates of both salivary glands and spleen and by higher serum levels of interleukin (IL)-2 and IL-4 (but not interferon-gamma and tumor necrosis factor-alpha) in infected versus noninfected rats. CONCLUSIONS: CMV infection of immunocompetent adult rats increases the neointimal response to vascular injury, suggesting that CMV may play a causal role in atherosclerosis/restenosis. Importantly, this CMV-induced response occurs even without the presence of virus in the vascular wall, suggesting that inflammatory and immune responses to infection of nonvascular tissues may contribute to the vascular response to injury.

The basis of molecular strategies for treating coronary restenosis after angioplasty.

Excessive smooth muscle cell proliferation significantly contributes to restenosis, which occurs in 25% to 50% of patients within 6 months of coronary angioplasty. Because successful treatment will probably depend on our acquiring a comprehensive knowledge of the molecular and cellular mechanisms involved, this report reviews 1) information relevant to the molecular and cellular mechanisms responsible for the smooth muscle cell(s) response to vascular injury, and 2) several molecular-based therapeutic strategies currently being explored as possible approaches to the control of restenosis, including recombinant DNA technology to target delivery of cytotoxic molecules to proliferating smooth muscle cell(s), antisense strategies to inhibit expression of gene products necessary for cell proliferation and gene therapy.

Signal transduction by reactive oxygen species in non-phagocytic cells.

A growing body of evidence suggests a potential role for oxygen-derived radicals such as superoxide anions and hydrogen peroxide as intracellular signaling molecules. Recently, progress has been made regarding the regulation of oxidant production in non-phagocytic cells. Significant gaps in understanding persist, however, especially in regard to the source(s) of oxidant production and the direct intracellular target(s) of oxygen radicals. Nonetheless, numerous recent studies have implicated a dynamic change in the intracellular redox state as an important determinant in a host of cellular decisions ranging from growth, to apoptosis, to cellular senescence.