Prevalence of XMRV nucleic acid and antibody in HIV-1-Infected men and in men at risk for HIV-1 Infection.

Xenotropic MLV-Related Virus (XMRV) was recently reported to be associated with prostate cancer and chronic fatigue syndrome (CFS). Infection was also reported in 3.7% of healthy individuals. These highly reported frequencies of infection prompted concerns about the possibility of a new, widespread retroviral epidemic. The Multicenter AIDS Cohort Study (MACS) provides an opportunity to assess the prevalence of XMRV infection and its association with HIV-1 infection among men who have sex with men. Reliable detection of XMRV infection requires the application of multiple diagnostic methods, including detection of human antibodies to XMRV and detection of XMRV nucleic acid. We, therefore, tested 332 patient plasma and PBMC samples obtained from recent visits in a subset of patients in the MACS cohort for XMRV antibodies using Abbott prototype ARCHITECT chemiluminescent immunoassays (CMIAs) and for XMRV RNA and proviral DNA using a XMRV single-copy qPCR assay (X-SCA). Although 9 of 332 (2.7%) samples showed low positive reactivity against a single antigen in the CMIA, none of these samples or matched controls were positive for plasma XMRV RNA or PBMC XMRV DNA by X-SCA. Thus, we found no evidence of XMRV infection among men in the MACS regardless of HIV-1 serostatus.

Genetic disruption of the Nrf2 compromises cell-cycle progression by impairing GSH-induced redox signaling.

Genetic disruption of Nrf2 greatly enhances susceptibility to prooxidant- and carcinogen-induced experimental models of various human disorders; but the mechanisms by which this transcription factor confers protection are unclear. Using Nrf2-proficient (Nrf2(+/+)) and Nrf2-deficient (Nrf2(-/-)) primary epithelial cultures as a model, we now show that Nrf2 deficiency leads to oxidative stress and DNA lesions, accompanied by impairment of cell-cycle progression, mainly G(2)/M-phase arrest. Both N-acetylcysteine and glutathione (GSH) supplementation ablated the DNA lesions and DNA damage-response pathways in Nrf2(-/-) cells; however only GSH could rescue the impaired colocalization of mitosis-promoting factors and the growth arrest. Akt activation was deregulated in Nrf2(-/-) cells, but GSH supplementation restored it. Inhibition of Akt signaling greatly diminished the GSH-induced Nrf2(-/-) cell proliferation and wild-type cell proliferation. GSH depletion impaired Akt signaling and mitosis-promoting factor colocalization in Nrf2(+/+) cells. Collectively, our findings uncover novel functions for Nrf2 in regulating oxidative stress-induced cell-cycle arrest, especially G(2)/M-checkpoint arrest, and proliferation, and GSH-regulated redox signaling and Akt are required for this process.

Stat4-dependent, T-bet-independent regulation of IL-10 in NK cells.

Interleukin-10 (IL-10) is intensely studied, yet little is known about the mechanisms that control IL-10 expression. We identified striking similarities between IL-10 and interferon-gamma (IFN-gamma) regulation in mouse natural killer (NK) cells. Like IFN-gamma, IL-10 expression is induced by IL-2 and IL-12 and IL-2+IL-12 stimulation is synergistic. Unlike IFN-gamma, neither IL-18 nor Ly-49D cross-linking induced IL-10 expression however. Additionally, the IL-12 homologs IL-23 and IL-27 also do not regulate NK cell-specific IL-10. We determined that a small population of NK cells accounts for IL-10 production. The induction of IL-10 by IL-2+IL-12 treatment in NK cells appears to be biphasic, with an initial burst of expression which diminishes by 12 h but spikes again at 18 h. We determined that much like IFN-gamma, Stat4 is largely required for IL-12-induced IL-10. Conversely, we observed normal induction of IL-10 in T-bet-deficient NK cells. We identified a Stat4-binding element in the fourth intron of the Il10 gene, which is completely conserved between mouse and human. This intronic Stat4 motif is within a conserved noncoding sequence, which is also a target for cytokine-induced histone acetylation. These findings highlight tissue- and receptor-specific IL-10 regulatory mechanisms, which may be part of an early feedback loop.

IFN-gamma: recent advances in understanding regulation of expression, biological functions, and clinical applications.

Interferon-gamma (IFN-gamma) is a key immunoregulatory protein that plays a major role in the host innate and adaptive immune response. Also known as type II interferon, IFN-gamma is a single-copy gene whose expression is regulated at multiple levels by the host. Transcription control is regulated through epigenetic mechanisms as well as the accessibility of chromatin and the binding of activating and inhibitory proteins to promoter and enhancer elements. Post-transcriptional control is mediated through mRNA localization and mRNA stability while post-translational control occurs through the activation of protein kinase R by the 5' portion of the mRNA, protein folding within the endoplasmic reticulum and the possible interaction of the mRNA with microRNAs. The biological effects of IFN-gamma are widespread, as almost every cell type is altered upon interaction with this protein. Thus it has become very apparent that IFN-gamma is a multipotent cytokine whose regulation and effects are complex and essential to host survival.

An IFNG SNP with an estrogen-like response element selectively enhances promoter expression in peripheral but not lamina propria T cells.

This study examines mucosa-specific regulatory pathways involved in modulation of interferon-gamma (IFN-gamma) in lamina propria T cells. Previous studies identified mucosa-specific CD2 cis-elements within the -204 to -108 bp IFNG promoter. Within this region, a single-site nucleotide polymorphism, -179G/T, imparts tumor necrosis factor-alpha stimulation of IFNG in peripheral blood lymphocytes, and is linked with accelerated AIDS progression. We discovered a putative estrogen response element (ERE) introduced by the -179T, which displays selective activation in peripheral blood mononuclear cells (PBMC) vs lamina propria mononuclear cells (LPMC). Transfection of PBMC with constructs containing the -179G or -179T site revealed CD2-mediated enhancement of the -179T compared to -179G allele, although, in LPMC, a similar level of expression was detected. Electrophoretic mobility shift assay (EMSA) analysis demonstrated CD2-mediated nucleoprotein binding to the -179T but not the -179G in PBMC. In LPMC, binding is constitutive to both -179G and -179T regions. Sequence and EMSA analysis suggests that the -179T allele creates an ERE-like binding site capable of binding recombinant estrogen receptor. Estrogen response element transactivation is enhanced by CD2 signaling, but inhibited by estrogen in PBMC but not in LPMC, although expression of estrogen receptor was similar. This is the first report to describe a potential molecular mechanism responsible for selectively controlling IFN-gamma production in LPMC.

A single nucleotide polymorphism in the proximal IFN-gamma promoter alters control of gene transcription.

Interferon-gamma (IFN-gamma) is an important cytokine that regulates cellular immune responses to intracellular pathogens and neoplasia. Regulation of IFN-gamma expression is stringently controlled at the transcriptional level. In this report we describe two novel single nucleotide polymorphisms (SNPs); one, at -179 in the promoter, occurs in 4% of African Americans. This SNP represents a guanidine to thymidine transition and creates a potential AP-1 binding element. Electrophoretic mobility shift analysis reveals a unique complex binding to an oligonucleotide containing the variant -179T but not to the -179G using nuclear extracts from human peripheral blood T cells. In reporter gene assays, T cell lines transfected with the variant -204(179T) IFN-gamma promoter show a six to 13-fold induction of luciferase activity in response to TNF-alpha over the common -204(179G) construct. The -179T allele identified in the proximal IFN-gamma promoter confers TNF-alpha inducibility and may prove important in human immune disorders and responsiveness to pathogens.

Interleukin-1 polymorphisms associated with increased risk of gastric cancer.

Helicobacter pylori infection is associated with a variety of clinical outcomes including gastric cancer and duodenal ulcer disease. The reasons for this variation are not clear, but the gastric physiological response is influenced by the severity and anatomical distribution of gastritis induced by H. pylori. Thus, individuals with gastritis predominantly localized to the antrum retain normal (or even high) acid secretion, whereas individuals with extensive corpus gastritis develop hypochlorhydria and gastric atrophy, which are presumptive precursors of gastric cancer. Here we report that interleukin-1 gene cluster polymorphisms suspected of enhancing production of interleukin-1-beta are associated with an increased risk of both hypochlorhydria induced by H. pylori and gastric cancer. Two of these polymorphism are in near-complete linkage disequilibrium and one is a TATA-box polymorphism that markedly affects DNA-protein interactions in vitro. The association with disease may be explained by the biological properties of interleukin-1-beta, which is an important pro-inflammatory cytokine and a powerful inhibitor of gastric acid secretion. Host genetic factors that affect interleukin-1-beta may determine why some individuals infected with H. pylori develop gastric cancer while others do not.

Polymorphisms of the human IFNG gene noncoding regions.

Interferon gamma (IFN-gamma) is a multifunctional cytokine that is essential in the development of Th1 cells and in cellular responses to a variety of intracellular pathogens including human immunodeficiency virus (HIV-1). We screened genomic DNA samples from a predominately Caucasian male population of HIV-infected and healthy donors for polymorphisms in the human IFNG gene from -777 to +5608 by single-stranded conformational polymorphism. Surprisingly, the proximal promoter (-777 to transcription start) is invariant as no polymorphisms were found in over 100 samples tested. However, further screening revealed polymorphisms in other regions of the gene including a single base insertion in a poly-T tract in the first intron, three single base pair substitutions in the third intron, and another single base pair substitution in the 3' untranslated region (UTR). Electrophoretic mobility shift assay was used to investigate whether these variants have altered DNA-binding abilities, since intronic enhancer elements have been reported for the IFNG gene. Oligonucleotides constructed for two third intron variants showed no difference in DNA-binding abilities as compared with wild-type sequences. However, the 3'UTR variant showed the formation of unique DNA-binding complexes to radiolabeled oligonucleotide probes as compared with the wild-type sequence. The influence of a CA-repeat microsatellite on AIDS disease progression in HIV-1 seroconverters was tested by a Cox proportional hazards model. There is no evidence of an association between alleles and infection with HIV-1 or progression to AIDS. We report an invariant proximal human IFNG promoter and the existence of multiple intronic variants and a potentially functional 3'UTR polymorphism.

Mucosa-specific targets for regulation of IFN-gamma expression: lamina propria T cells use different cis-elements than peripheral blood T cells to regulate transactivation of IFN-gamma expression.

Activation of lamina propria (LP) T cells via the CD2 pathway enhances IFN-gamma (IFN-gamma) secretion with further enhancement after CD28 coligation. The molecular mechanisms regulating IFN-gamma expression in LP T cells remain unknown. Previous studies in PBL and T cell lines identified cis- and trans-regulatory elements in TCR-mediated expression of IFN-gamma. This study examines CD2 and PMA/ionophore-responsive IFN-gamma promoter elements. Activation of LPMC via CD2-induced IFN-gamma secretion and a parallel up-regulation of mRNA expression. CD28 coligation enhanced mRNA stability without up-regulating transcription as measured by nuclear run-on. Transfection of a -2.7-kb IFN-gamma promoter-reporter construct into PBL and LP mononuclear cells (LPMC) revealed significant promoter activity after CD2 activation, with additional transactivation after CD2/CD28 costimulation in PBL, but not in LPMC. Functional analysis using truncated promoter fragments identified distinct cis-regulatory regions selectively transactivating IFN-gamma expression in PBL compared with LPMC. In PBL, CD2 activation elements reside within the -108- to +64-bp region. However, in LPMC the upstream region between -204 and -108 bp was essential. Transfection of the proximal and distal AP-1-binding elements, as well as TRE/AP-1 constructs, revealed functional activation of AP-1 subsequent to CD2 signaling, with activation critical in PBL but diminished in LPMC. Electromobility shift analysis using oligonucleotides encompassing the proximal, distal, and BED/AP-1-binding regions failed to demonstrate selective transactivation after CD2 signaling of LPMC. This report provides evidence that activation of LPMC results in transactivation of multiple promoter elements regulating IFN-gamma expression distinct from those in PBL.