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3.  Interferon Alpha in Systemic Lupus Erythematosus 
The pleiotropic cytokine interferon alpha is involved in multiple aspects of lupus etiology and pathogenesis. Interferon alpha is important under normal circumstances for antiviral responses and immune activation. However, heightened levels of serum interferon alpha and expression of interferon response genes are common in lupus patients. Lupus-associated autoantibodies can drive the production of interferon alpha and heightened levels of interferon interfere with immune regulation. Several genes in the pathways leading to interferon production or signaling are associated with risk for lupus. Clinical and cellular manifestations of excess interferon alpha in lupus combined with the genetic risk factors associated with interferon make this cytokine a rare bridge between genetic risk and phenotypic effects. Interferon alpha influences the clinical picture of lupus and may represent a therapeutic target. This paper provides an overview of the cellular, genetic, and clinical aspects of interferon alpha in lupus.
doi:10.1155/2010/948364
PMCID: PMC2896914  PMID: 20652065
4.  Four Promoters of IRF5 Respond Distinctly to Stimuli and are Affected by Autoimmune-Risk Polymorphisms 
Introduction: Autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis, and multiple sclerosis affect millions of people worldwide. Interferon regulatory factor 5 (IRF5) contains polymorphisms associated with these autoimmune diseases. Two of these functional polymorphisms are found upstream of the IRF5 gene. rs2004640, which is a single nucleotide polymorphism and the CGGGG insertion/deletion (indel) were studied. IRF5 uses four different promoters for its four first exons: 1A, 1B, 1C, and 1D. Each promoter was analyzed, including functional differences due to the autoimmune-risk polymorphisms.
Results: IRF5 promoters were analyzed using ChIP-Seq data (ENCODE database) and the FactorBook database to define transcription factor binding sites. To verify promoter activity, the promoters were cloned into luciferase plasmids. Each construct exhibited luciferase activity. Exons 1A and 1D contain putative PU.1 and NFkB binding sites. Imiquimod, a Toll-like receptor 7 (TLR7) ligand, was used to activate these transcription factors. IRF5 levels were doubled after imiquimod treatment (p < 0.001), with specific increases in the 1A promoter (2.2-fold, p = 0.03) and 1D promoter (2.8-fold, p = 0.03). A putative binding site for p53, which affects apoptosis, was found in the promoter for exon 1B. However, site-directed mutagenesis of the p53 site showed no effect in a reporter assay.
Conclusion: The IRF5 exon 1B promoter has been characterized, and the responses of each IRF5 promoter to TLR7 stimulation have been determined. Changes in promoter activity and gene expression are likely due to specific and distinct transcription factors that bind to each promoter. Since high expression of IRF5 contributes to the development of autoimmune disease, understanding the source of increased IRF5 levels is key to understanding autoimmune etiology.
doi:10.3389/fimmu.2013.00360
PMCID: PMC3819785  PMID: 24223576
IRF5; alternative promoters; autoimmune disease risk; interferon; systemic lupus erythematosus
5.  Effects of IRF5 Lupus Risk Haplotype on Pathways Predicted to Influence B Cell Functions 
Both genetic and environmental interactions affect systemic lupus erythematosus (SLE) development and pathogenesis. One known genetic factor associated with lupus is a haplotype of the interferon regulatory factor 5 (IRF5) gene. Analysis of global gene expression microarray data using gene set enrichment analysis identified multiple interferon- and inflammation-related gene sets significantly overrepresented in cells with the risk haplotype. Pathway analysis using expressed genes from the significant gene sets impacted by the IRF5 risk haplotype confirmed significant correlation with the interferon pathway, Toll-like receptor pathway, and the B-cell receptor pathway. SLE patients with the IRF5 risk haplotype have a heightened interferon signature, even in an unstimulated state (P = 0.011), while patients with the IRF5 protective haplotype have a B cell interferon signature similar to that of controls. These results identify multiple genes in functionally significant pathways which are affected by IRF5 genotype. They also establish the IRF5 risk haplotype as a key determinant of not only the interferon response, but also other B-cell pathways involved in SLE.
doi:10.1155/2012/594056
PMCID: PMC3304673  PMID: 22500098
6.  Characterization of herpes simplex virus clinical isolate Y3369 as a glycoprotein G variant and its bearing on virus typing 
Virology Journal  2011;8:290.
Background
Herpes simplex viruses exist as two major serotypes, type 1 (HSV-1) and type 2 (HSV-2). Determination of type, either HSV-1 or HSV-2, is important in accurate diagnosis and clinical control of transmission. Several tests are available for typing HSV, including a monoclonal antibody specific for glycoprotein G and several PCR assays.
Findings
A clinical isolate was identified as herpes simplex virus, but tested negative for both HSV-1 and HSV-2 antigens using type-specific monoclonal antibody assays. The isolate was determined to be HSV-1 by PCR analysis. A mutation which likely caused the monoclonal antibody non-reactivity was found in glycoprotein G. Phylogenetic analysis revealed two groups of HSV, one with the mutation and one without. Three population studies examining mutations in HSV-1 glycoprotein G were analyzed by chi-squared test. To this point, the epitope which the monoclonal antibody recognizes was only found in HSV-1 isolates from human European populations (p < 0.0001).
Conclusions
These findings suggest that the PCR-based methods for HSV typing may be more useful than the standard monoclonal antibody test in areas of the world where the variant in glycoprotein G is more prevalent.
doi:10.1186/1743-422X-8-290
PMCID: PMC3118968  PMID: 21658271
Herpes Simplex Virus; serotyping; glycoprotein G
7.  Parvovirus B19 Nonstructural Protein-Induced Damage of Cellular DNA and Resultant Apoptosis 
Parvovirus B19 is a widespread virus with diverse clinical presentations. The viral nonstructural protein, NS1, binds to and cleaves the viral genome, and induces apoptosis when transfected into nonpermissive cells, such as hepatocytes. We hypothesized that the cytotoxicity of NS1 in such cells results from chromosomal DNA damage caused by the DNA-nicking and DNA-attaching activities of NS1. Upon testing this hypothesis, we found that NS1 covalently binds to cellular DNA and is modified by PARP, an enzyme involved in repairing single-stranded DNA nicks. We furthermore discovered that the DNA nick repair pathway initiated by poly(ADPribose)polymerase and the DNA repair pathways initiated by ATM/ATR are necessary for efficient apoptosis resulting from NS1 expression.
PMCID: PMC3030141  PMID: 21278893
Parvovirus B19; DNA damage and repair; fulminant liver failure; apoptosis; autoantibody; systemic lupus erythematosus
8.  Lupus-like autoantibody development in rabbits and mice after immunization with EBNA-1 fragments 
Journal of autoimmunity  2008;31(4):362-371.
Epstein-Barr virus has been implicated in the etiology of systemic lupus erythematosus (SLE) through serologic and immunologic studies. A potential mechanism for this influence is through molecular mimicry. The EBV nuclear antigen EBNA-1 contains a region, PPPGRRP, with considerable homology to the initial sequence targeted by antibodies in Sm B’ autoimmunity, PPPGMRPP. This study examined whether immunization of rabbits and mice with peptides containing the PPPGRRP sequence from EBNA-1 constructed on a poly-lysine backbone was able to drive the development of autoantibodies against the Smith antigen (Sm) and the related antigenic complex, the U1 nuclear ribonucleoproteins (nRNP). PPPGRRP immunization, and immunization with an EBNA-1 fragment containing PPPGRRP, led to autoantibodies in both rabbits and mice at high frequency (83% of rabbits and 43% of mice). Five out of six immunized rabbits developed either leucopenia or lymphopenia or both. The fine specificity of antibody binding against the lupus-associated autoantigens Sm B’, nRNP A, and nRNP C after immunization with the EBNA-1-derived peptides was very similar to the early antibody binding patterns against these proteins in human SLE. This similarity, as well as the prevalence of autoimmunity after immunization with these peptides, identifies PPPGRRP as a strong candidate for molecular mimicry in SLE etiology.
doi:10.1016/j.jaut.2008.08.007
PMCID: PMC2852321  PMID: 18849143
autoantibodies; molecular mimicry; systemic lupus erythematosus; Epstein-Barr virus
9.  Early Targets of nRNP Humoral Autoimmunity in Human Systemic Lupus Erythematosus 
Arthritis and rheumatism  2009;60(3):848-859.
Objective:
The U1 small nuclear ribonucleoproteins (nRNPs) are common targets of autoantibodies in lupus and other autoimmune diseases. However, the etiology and progression of autoimmune responses directed against these antigens are not well understood. Using a unique collection of serial human samples from before and after nRNP antibody development, we investigated early humoral events in the development of anti-nRNP autoimmunity.
Methods:
Lupus patients with sera available from both before and after nRNP antibody precipitin development were identified from the Oklahoma Clinical Immunology Serum Repository. Antibodies in the serial samples were analyzed by ELISA, Western blotting, solid-phase epitope mapping and competition assays.
Results:
The first detected nRNP antibodies targeted 6 common initial epitopes in nRNP A, 2 in nRNP C and 9 in nRNP 70K. The initial epitopes of nRNP A and nRNP C were significantly enriched for proline (p=0.0004, p=0.048) and shared up to 95% sequence homology. The initial nRNP 70K humoral epitopes differed from nRNP A and C. The initial antibodies to nRNP A and nRNP C were cross-reactive with the Sm B′-derived peptide PPPGMRPP. Antibody binding against all three nRNP subunits diversified significantly over time.
Conclusions:
nRNP A and nRNP C autoantibodies initially targeted restricted, proline-rich motifs. Antibody binding subsequently spread to other epitopes. The similarity and cross-reactivity between the initial targets of nRNP and Sm autoantibodies identifies a likely commonality in etiology and a focal point for intermolecular epitope spreading.
doi:10.1002/art.24306
PMCID: PMC2653589  PMID: 19248110
10.  Aberrant Epstein–Barr viral infection in systemic lupus erythematosus☆ 
Autoimmunity reviews  2009;8(4):337.
Serologic association, cross-reactivity of select EBV-specific antibodies with SLE autoantigens, SLE-like autoimmunity after immunization with EBV peptides, increased EB viral load in SLE patients, and SLE-specific alterations in EBV humoral and cellular immunity implicate Epstein–Barr virus (EBV) in the development of systemic lupus erythematosus (SLE). To investigate SLE-specific differences in EBV gene expression, levels of eight EBV genes were compared between SLE patients and controls by using both ex vivo-infected and un-manipulated peripheral blood mononuclear cells (PBMCs). Expression levels of mRNA were significantly greater by Wilcoxen signed rank test in the ex vivo-infected SLE patient-derived cells for 4 of 8 EBV genes, including BLLF1, 3.2-fold (p<0.004); LMP-2, 1.7-fold (p<0.008); EBNA-1, 1.7-fold (p<0.01); and BcRF1, a proposed DNA binding protein, 1.7-fold (p<0.02). The frequency of LMP-1 gene expression was significantly greater by Chi square analysis in the peripheral blood from SLE patients than controls (44% of patients, 10% of controls p<0.05). PBMCs from SLE patients had greater expression of latent genes as well as increased expression of both latent and lytic genes after infection, suggesting that EBV may participate in SLE etiology through several mechanisms. Such altered infection patterns may contribute to the increased levels of EBV and the molecular mimicry seen in sera from SLE patients.
doi:10.1016/j.autrev.2008.12.008
PMCID: PMC2822456  PMID: 19167523
Systemic lupus erythematosus; Epstein–Barr virus; Gene expression; Latency
11.  Apoptosis of Liver-Derived Cells Induced by Parvovirus B19 Nonstructural Protein 
Journal of Virology  2006;80(8):4114-4121.
Parvovirus B19 has been implicated in some cases of acute fulminant non-A, non-B, non-C, non-G liver failure. Our laboratory previously demonstrated that B19 infection of hepatocytes induces apoptosis and that the B19 viral nonstructural protein, NS1, may play a critical role. To study the involvement of NS1 in apoptosis of liver cells, we generated a fusion protein of NS1 with enhanced green fluorescent protein (eGFP) in a system allowing for inducible gene expression. Transfection of the liver-derived cell line HepG2 with the eGFP/NS1 vector allowed expression of the fusion protein, which was visualized by fluorescence microscopy and demonstrated by immunoblotting. The fusion protein localized to discrete domains in the nucleus. Transfection of HepG2 cells with the eGFP/NS1 vector led to apoptosis of 35% of transfected cells, a sevenfold increase over cells transfected with the parent eGFP expression vector. Mutation of the eGFP/NS1 vector to eliminate the nucleoside triphosphate-binding site of NS1 significantly decreased apoptosis, as did treatment of transfected cells with inhibitors of caspase 3 or 9. Neutralization of tumor necrosis factor alpha or Fas ligand had no effect on apoptosis. These results demonstrate that NS1 is sufficient to induce apoptosis in liver-derived cells and that it does so through the initiation of an intrinsic caspase pathway.
doi:10.1128/JVI.80.8.4114-4121.2006
PMCID: PMC1440431  PMID: 16571827
12.  Parvovirus B19-Induced Apoptosis of Hepatocytes 
Journal of Virology  2004;78(14):7775-7783.
Parvovirus B19 (B19 virus) can persist in multiple tissues and has been implicated in a variety of diseases, including acute fulminant liver failure. The mechanism by which B19 virus induces liver failure remains unknown. Hepatocytes are nonpermissive for B19 virus replication. We previously reported that acute fulminant liver failure associated with B19 virus infection was characterized by hepatocellular dropout. We inoculated both primary hepatocytes and the hepatocellular carcinoma cell line Hep G2 with B19 virus and assayed for apoptosis by using annexin V staining. Reverse transcriptase PCR analysis and immunofluorescence demonstrated that B19 virus was able to infect the cells and produce its nonstructural protein but little or no structural capsid protein. Infection with B19 virus induced means of 28% of Hep G2 cells and 10% of primary hepatocytes to undergo apoptosis, which were four- and threefold increases, respectively, over background levels. Analysis of caspase involvement showed that B19 virus-inoculated cultures had a significant increase in the number of cells with active caspase 3. Inhibition studies demonstrated that caspases 3 and 9, but not caspase 8, are required for B19 virus-induced apoptosis.
doi:10.1128/JVI.78.14.7775-7783.2004
PMCID: PMC434113  PMID: 15220451

Results 1-12 (12)