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1.  Assessment of gene expression in peripheral blood using RNAseq before and after weight restoration in anorexia nervosa 
Psychiatry research  2013;210(1):287-293.
We examined gene expression in the blood of six females with anorexia nervosa (AN) before and after weight restoration using RNAseq. AN cases (aged 19-39) completed clinical assessments and had blood drawn for RNA at hospital admission (T1, < ~75% ideal body weight, IBW) and again at discharge (T2, ≥ ~85% IBW). To examine the relationship between weight restoration and differential gene expression, normalized gene expression levels were analyzed using a paired design. We found 564 genes whose expression was nominally significantly different following weight restoration (p < 0.01, 231 increased and 333 decreased). With a more stringent significance threshold (false discovery rate q < 0.05), 67 genes met criteria for differential expression. Of the top 20 genes, CYP11A1, C16orf11, LINC00235, and CPA3 were down-regulated more than two-fold after weight restoration while multiple olfactory receptor genes (OR52J3, OR51L1, OR51A4, OR51A2) were up-regulated more than two-fold after weight restoration. Pathway analysis revealed up-regulation of two broad pathways with largely overlapping genes, one related to protein secretion and signaling and the other associated with defense response to bacterial regulation. Although results are preliminary secondary to a small sample size, these data provide initial evidence of transcriptional alterations during weight restoration in AN.
PMCID: PMC3805820  PMID: 23778302
biomarkers; renourishment; transcriptome; eating disorder; genetic; genome
2.  Gout, tophi and the wonders of NETs 
PMCID: PMC4149263
3.  Effects of Cartilage Impact with and without Fracture on Chondrocyte Viability and the Release of Inflammatory Markers 
Post-traumatic arthritis (PTA) frequently develops after intra-articular fracture of weight bearing joints. Loss of cartilage viability and post-injury inflammation have both been implicated as possible contributing factors to PTA progression. In order to further investigate chondrocyte response to impact and fracture, we have developed a blunt impact model applying 70%, 80% or 90% surface-to-surface compressive strain with or without induction of an articular fracture in a cartilage explant model. Following mechanical loading, chondrocyte viability and apoptosis were assessed. Culture media were evaluated for the release of double-stranded DNA (dsDNA) and immunostimulatory activity via nuclear factor kappa B (NF-κB) activity in Toll-like receptor-expressing Ramos-Blue reporter cells. High compressive strains, with or without articular fracture, resulted in significantly reduced chondrocyte viability. Blunt impact at 70% strain induced a loss in viability over time through a combination of apoptosis and necrosis, whereas blunt impact above 80% strain caused predominantly necrosis. In the fracture model, a high level of primarily necrotic chondrocyte death occurred along the fracture edges. At sites away from the fracture, viability was not significantly different than controls. Interestingly, both dsDNA release and NF-κB activity in Ramos-Blue cells increased with blunt impact, but was only significantly increased in the media from fractured cores. This study indicates that the mechanism of trauma determines the type of chondrocyte death as well as the potential for post-injury inflammation.
PMCID: PMC3966619  PMID: 23620164
posttraumatic arthritis; chondrocyte viability; apoptosis; intra-articular fracture; inflammation
4.  Tumor necrosis factor: is it time to change the name? 
PMCID: PMC4060199  PMID: 25166863
5.  The Expression of HMGB1 on Microparticles Released during Cell Activation and Cell Death In Vitro and In Vivo 
Molecular Medicine  2014;20(1):158-163.
High mobility group box protein 1 (HMGB1) is a nonhistone nuclear protein that is a prototypic alarmin that can stimulate innate immunity and drive the pathogenesis of a wide range of inflammatory diseases. While HMGB1 can be released from both activated and dying cells, its biochemical and immunological properties differ depending on the release mechanism, resulting from redox changes and posttranslational modifications including acetylation. In addition to release of HMGB1, cell death is associated with the release of microparticles. Microparticles are small membrane-bound vesicles that contain cytoplasmic, nuclear and membrane components. Like HMGB1, microparticles display immunological activity and levels are elevated in diseases characterized by inflammation and vasculopathy. While studies have addressed the immunological effects of HMGB1 and microparticles independently, HMGB1, like other nuclear molecules, is a component of microparticles. Evidence for the physical association of HMGB1 comes from Western blot analysis of microparticles derived from RAW 264.7 macrophage cells stimulated by lipopolysaccharide (LPS) or induced to undergo apoptosis by treatment with etoposide or staurosporine in vitro. Analysis of microparticles in the blood of healthy volunteers receiving LPS shows the presence of HMGB1 as assessed by flow cytometry. Together, these findings indicate that HMGB1 can be a component of microparticles and may contribute to their activities. Furthermore, particle HMGB1 may represent a useful biomarker for in vivo events that may not be reflected by measurement of the total amount of HMGB1 in the blood.
PMCID: PMC4002850  PMID: 24618884
6.  The Role of HMGB1 in the Pathogenesis of Inflammatory and Autoimmune Diseases 
Molecular Medicine  2014;20(1):138-146.
High-mobility group box 1 (HMGB1) protein is a highly abundant protein that can promote the pathogenesis of inflammatory and autoimmune diseases once it is in an extracellular location. This translocation can occur with immune cell activation as well as cell death, with the conditions for release associated with the expression of different isoforms. These isoforms result from post-translational modifications, with the redox states of three cysteines at positions 23, 45 and 106 critical for activity. Depending on the redox states of these residues, HMGB1 can induce cytokine production via toll-like receptor 4 (TLR4) or promote chemotaxis by binding the chemokine CXCL12 for stimulation via CXCR4. Fully oxidized HMGB1 is inactive. During the course of inflammatory disease, HMGB1 can therefore play a dynamic role depending on its redox state. As a mechanism to generate alarmins, cell death is an important source of HMGB1, although each major cell death form (necrosis, apoptosis, pyroptosis and NETosis) can lead to different isoforms of HMGB1 and variable levels of association of HMGB1 with nucleosomes. The association of HMGB1 with nucleosomes may contribute to the pathogenesis of systemic lupus erythematosus by producing nuclear material whose immunological properties are enhanced by the presence of an alarmin. Since HMGB1 levels in blood or tissue are elevated in many inflammatory and autoimmune diseases, this molecule can serve as a unique biomarker as well as represent a target of novel therapies to block its various activities.
PMCID: PMC3966993  PMID: 24531836
7.  The Effects of Smoking on Levels of Endothelial Progenitor Cells and Microparticles in the Blood of Healthy Volunteers 
PLoS ONE  2014;9(2):e90314.
Cigarette smoking, both active and passive, is one of the leading causes of morbidity and mortality in cardiovascular disease. To assess the impact of brief smoking on the vasculature, we determined levels of circulating endothelial progenitor cells (EPCs) and circulating microparticles (MPs) following the smoking of one cigarette by young, healthy intermittent smokers.
Materials and Methods
12 healthy volunteers were randomized to either smoking or not smoking in a crossover fashion. Blood sampling was performed at baseline, 1, 4 and 24 hours following smoking/not smoking. The numbers of EPCs and MPs were determined by flow cytometry. MPs were measured from platelets, leukocytes and endothelial cells. Moreover, MPs were also labelled with anti-HMGB1 and SYTO 13 to assess the content of nuclear molecules.
Active smoking of one cigarette caused an immediate and significant increase in the numbers of circulating EPCs and MPs of platelet-, endothelial- and leukocyte origin. Levels of MPs containing nuclear molecules were increased, of which the majority were positive for CD41 and CD45 (platelet- and leukocyte origin). CD144 (VE-cadherin) or HMGB1 release did not significantly change during active smoking.
Brief active smoking of one cigarette generated an acute release of EPC and MPs, of which the latter contained nuclear matter. Together, these results demonstrate acute effects of cigarette smoke on endothelial, platelet and leukocyte function as well as injury to the vascular wall.
PMCID: PMC3938677  PMID: 24587320
8.  The Choosing Wisely initiative: Does it have your back? 
PMCID: PMC3978556  PMID: 23998494
9.  The role of microparticles in the generation of immune complexes in murine lupus 
Systemic lupus erythematosus is a systemic inflammatory disease characterized by antibodies to nuclear molecules in association with immune complex deposition. As shown previously, microparticles (MPs), which are small membrane-bound vesicles released from dying and activated cells, contain nucleic acids and can form immune complexes found in patient blood. To assess the role of MPs in murine lupus, we used flow cytometry to measure the presence of MPs with bound IgG in the blood of MRL-lpr/lpr and NZB/W mice. These studies showed much higher numbers of MPs with bound IgG in the blood of MRL lpr/lpr compared to NZB/W mice. Furthermore, these studies showed that antibodies from MRL-lpr/lpr mice bound better to MPs from apoptotic cells than those from NZB/W mice. Together, these studies indicate important differences in the serological features of the two strains as reflected by the capacity of antibodies to bind to MPs.
PMCID: PMC3551532  PMID: 23159786
autoimmunity; animal models; apoptosis; molecular immunology
10.  The 2013 ACR Meeting: Mad Macs at the Marina 
PMCID: PMC3978445  PMID: 24364924
11.  The effectors of innate immunity: DAMPs, DAMEs, or DIMEs? 
PMCID: PMC3978486  PMID: 24172019
12.  Microparticles as mediators and biomarkers of rheumatic disease 
Rheumatology (Oxford, England)  2012;51(10):1737-1746.
Microparticles (MPs) are small membrane-bound vesicles that arise from activated and dying cells and enter the blood to display pro-inflammatory and pro-thrombotic activities. MPs are 0.1–1.0 μm in size and incorporate nuclear, cytoplasmic and membrane molecules as they detach from cells. This process can occur with cell activation as well as cell death, with particles likely corresponding to blebs that form on the cell surface during apoptosis. To measure particle expression, flow cytometry allows determination of particle numbers based on size as well as surface markers that denote the cell of origin; platelet MPs are usually the most abundant type in blood. As shown in in vitro and in vivo systems, MPs can promote inflammation and thrombosis resulting from their content of cytokines like IL-1 and pro-coagulant molecules like tissue factor. Certain particle types can be anti-inflammatory, however, suggesting a range of immunomodulatory activities depending on the cell of origin. Studies on patients with a wide range of rheumatic disease show increased MP numbers in blood, with platelet and endothelial particles associated with vascular manifestations; increased numbers of particles also occur in the joint fluid where they may drive cytokine production and activate synoviocytes. In autoimmune diseases such as SLE and RA, MPs may also contribute to disease pathogenesis by the formation of immune complexes. MPs thus represent novel subcellular structures that can impact on the pathogenesis of rheumatic disease and serve as biomarkers of underlying cellular disturbances.
PMCID: PMC3448881  PMID: 22403183
microparticles; inflammation; thrombosis; rheumatic disease; apoptosis; autoimmunity; DNA; RNA
13.  The butterfly rash of lupus: An example of aposematism? 
PMCID: PMC3672680  PMID: 23425372
14.  The Origin and Properties of Extracellular DNA: From PAMP to DAMP 
DNA is a polymeric macromolecule whose biological activities depend on location as well as binding to associated molecules. Inside the cell, DNA is the source of genetic information and binds histones to form nucleosomes. DNA can exit the cell, however, to enter the extracellular space primarily during cell death, either apoptosis or necrosis, as well as NETosis. While bacterial DNA is a potent immune stimulant by virtue of its CpG motifs, mammalian DNA, which is ordinarily inactive, can acquire activity by associating with nuclear, cytoplasmic and serum proteins which promote its uptake into cells to stimulate internal DNA sensors, including toll-like receptor 9. Among these proteins, anti-DNA autoantibodies can form immune complexes with DNA to stimulate plasmacytoid dendritic cells to produce type 1 interferon. Together, these findings suggest that the immune properties of DNA are mutable and diverse, reflecting its context and the array of attached molecules.
PMCID: PMC3724456  PMID: 22659033
DNA; anti-DNA; systemic lupus erythematosus; apoptosis; immune complexes; HMGB1
15.  The Induction of HMGB1 Release from RAW 264.7 Cells by Transfected DNA 
Molecular immunology  2007;45(7):2038-2044.
High mobility group protein 1 (HMGB1) is a non-histone nuclear protein that can activate innate immunity when in an extracellular location. As shown in in vitro studies, while polyinosinic-polycytidylic acid [poly (I:C)] and LPS, TLR3 and TLR4 ligands respectively, can induce HMGB1 release from macrophages, CpG DNA, a TLR 9 ligand, does not. Since DNA displays distinct immunostimulatory activity when transfected into cells, we investigated whether transfected DNA can induce HMGB1 release from macrophages. In these experiments, using RAW 264.7 cells as model, we show that DNA, either natural DNA or synthetic oligonucleotides, can induce HMGB1 release when used to stimulate cells with the transfection reagent Lipofectamine 2000®; release occurred irrespective of the intrinsic activity of the DNA. The induction of HMGB1 release by transfected DNA was dependent on IFN-β as shown by the inhibitory effects of an antibody. In addition, JNK activation mediated HMGB1 release induced by a transfected phosphorothioate oligonucleotide but not by transfected natural DNA. Together, these findings indicate that transfected DNA can stimulate macrophages to release HMGB1 under conditions in which free DNA is inactive and suggest a role of DNA in inducing inflammation when bound to molecules that influence its entry into cells.
PMCID: PMC3724460  PMID: 18031817
DNA; HMGB1; transfection; inflammation
16.  Microparticles as Autoantigens: Making Immune Complexes Big 
Arthritis and rheumatism  2012;64(4):958-961.
PMCID: PMC3724462  PMID: 22237935
autoantibodies; autoantigens; immune complexes; microparticles; systemic lupus erythematosus
17.  Cell Death in the Pathogenesis of Immune-Mediated Diseases: The Role of HMGB1 and DAMP-PAMP Complexes 
Swiss medical weekly  2011;141:w13256.
Cell death is a ubiquitous process whose immunological consequences can influence the course of infectious, autoimmune and inflammatory diseases. While cell death was long dichotomized in terms of apoptosis and necrosis, other forms of death can occur and vary in their capacity to stimulate as well as inhibit inflammation. The pro-inflammatory activity of dead cells results from a wide variety of intracellular molecules that are released as cell permeability breaks during death. These molecules have been termed as DAMPs (damage associated molecular patterns) or alarmins. Among these DAMPs, HMGB1, a non-histone nuclear protein, serves as the prototype. Although HMGB1 was originally thought to act alone as a cytokine, recent studies suggest that its immunological effects result from complexes of HMGB1 with either other DAMPs or with PAMPs (pathogen associated molecular patterns). Together, studies on the role HMGB1 in pathogenesis suggest that the formation of extracellular complexes is an important mechanism for generating pro-inflammatory signals during cell death and therefore a potential target of new therapy.
PMCID: PMC3724463  PMID: 21877298
apoptosis; necrosis; alarmin; inflammation; HMGB1
18.  The extracellular release of DNA and HMGB1 from Jurkat T cells during in vitro necrotic cell death 
Innate immunity  2012;18(5):727-737.
In innate immunity, dead and dying cells release internal constituents that can serve as DAMPs (damage-associated molecular patterns) or alarmins. This release occurs more abundantly during necrosis than apoptosis and may account for the differences in the immunological properties of these death forms. To elucidate DAMP release in necrosis, we have compared the levels of two nuclear molecules (DNA and HMGB1, a non-histone protein with alarmin activity) in the media following necrosis of Jurkat T cells by freeze-thawing, ethanol, heat or hydrogen peroxide. In our experiments, DNA release was measured by fluorimetry with the dye PicoGreen, while HMGB1 was measured by Western blotting. As results of this study show, each form of necrosis is associated with a distinct pattern of DNA and HMGB1 release with respect to kinetics and amounts. Of these, freeze-thawing produced the highest and most rapid increase in HMGB1 and DNA levels although the released DNA was subject to nuclease digestion; in addition, freeze-thawing led to the production of particles measured by flow cytometry. Together, these results indicate that experimental necrosis leads to diverse patterns of nuclear molecule release which could affect their immunological activity.
PMCID: PMC3724467  PMID: 22344226
alarmin; DNA; HMGB1; immune activation; necrosis
19.  Microparticles as a source of extracellular DNA 
Immunologic research  2011;49(0):227-234.
Microparticles are small membrane-bound vesicles that display pro-inflammatory and pro-thrombotic activities important in the pathogenesis of a wide variety of diseases. These particles are released from activated and dying cells and incorporate nuclear and cytoplasmic molecules for extracellular export. Of these molecules, DNA is a central autoantigen in systemic lupus erythematosus (SLE). As studies in our laboratory show, DNA occurs prominently in microparticles, translocating into these structures during apoptotic cell death. This DNA is antigenically active and can bind to lupus anti-DNA autoantibodies. These findings suggest that microparticles are an important source of extracellular DNA to serve as an autoantigen and autoadjuvant in SLE.
PMCID: PMC3724471  PMID: 21132466
Microparticles; DNA; Apoptosis; Systemic lupus erythematosus; Innate immunity
20.  Advances in the treatment of inflammatory arthritis 
The inflammatory arthritides are a diverse group of conditions characterised by joint inflammation which can lead to pain, deformity and disability. Of these diseases, rheumatoid arthritis (RA) and spondyloarthritis are two of the most common. While the clinical and demographic features of these diseases differ, the central role of inflammation in their pathogenesis has allowed the development of highly effective treatment strategies with wide applicability. These strategies include the use of biological agents which target the cytokine tumour necrosis factor (TNF), a key mediator of inflammation. With the advent of effective agents, therapy has become more aggressive, reducing disease activity and allowing, at least in RA, remission in many patients. While the array of available effective treatments is extensive, the use of objective measures of disease activity can guide treatment decisions (treat to target) and lead to improved outcomes.
PMCID: PMC3461328  PMID: 22794097
Rheumatoid arthritis; Spondyloarthritis; Ankylosing spondylitis
22.  The LE cell: crime scene or crime stopper? 
PMCID: PMC3446528  PMID: 22738266
23.  HMGB1 and Microparticles as Mediators of the Immune Response to Cell Death 
Antioxidants & Redox Signaling  2011;15(8):2209-2219.
In a wide variety of diseases, cell death represents both an outcome and an important step in pathogenesis. This duality occurs because cell death leads to the extracellular release of molecules and structures that can potently induce the innate immune system. These mediators include the alarmins which are endogenous cellular constituents that exit activated or dying cells to stimulate toll-like receptors (TLRs) as well as non-TLR receptors. Of alarmins, the nonhistone protein HMGB1 is the prototype. Like DNA and RNA, HMGB1 can translocate from cells as they die. The activity of HMGB1 may reflect its interaction with other molecules such as LPS, DNA, and cytokines. In addition to alarmins, dead and dying cells can release subcellular organelles called microparticles that contain cytoplasmic and nuclear constituents, including DNA and RNA. These particles can impact on many cell types to induce inflammation. The release of HMGB1 and microparticles shows important similarities, occurring with cell death as well as stimulation of certain but not all TLRs. Furthermore, nitric oxide can induce the release of both. These observations suggest that the products of dead cells can serve as important mediators to drive immune responses and promote inflammation and autoreactivity. Antioxid. Redox Signal. 15, 2209–2219.
PMCID: PMC3166182  PMID: 21194388
24.  HMGB1: a smoking gun in lupus nephritis? 
High-mobility group box 1 protein (HMGB1) is a prototypic alarmin that is released from activated and dying cells. Because of its proinflammatory activities, HMGB1 could mediate key events in the pathogenesis of systemic lupus erythematosus, a possibility supported by elevations of HMGB1 in patient blood and increased expression in renal biopsies. The biology of HMGB1 is complicated, however, and its activity is dependent on redox state as well as binding to other molecules such as cytokines. Defining more precisely the role of HMGB1 in lupus will require treatment studies to block the activity of this alarmin in animal models and ultimately patients.
PMCID: PMC3446408  PMID: 22423653
25.  Identification of Novel Innate Immune Genes by Transcriptional Profiling of Macrophages Stimulated with TLR Ligands 
Molecular immunology  2011;48(15-16):1886-1895.
Toll-like receptors (TLRs) are key receptors in innate immunity and trigger responses following interaction with pathogen-associated molecular patterns (PAMPs). TLR3, TLR4 and TLR9 recognize double stranded RNA, lipopolysaccharide (LPS) and CpG DNA, respectively. These receptors differ importantly in downstream adaptor molecules. TLR4 signals through MyD88 and TRIF; in contrast, the TLR3 pathway involves only TRIF while TLR9 signals solely through MyD88. To determine how differences in downstream signaling could influence gene expression in innate immunity, gene expression patterns were determined for the RAW264.7 macrophage cell line stimulated with LPS, poly (I:C), or CpG DNA. Gene expression profiles 6 and 24 hrs post-stimulation were analyzed to determine genes, pathways and transcriptional networks induced. As these experiments showed, the number and extent of genes expressed varied with stimulus. LPS and poly (I:C) induced an abundant array of genes in RAW264.7 cells at 6 hrs and 24 hrs following treatment while CpG DNA induced many fewer. By analyzing data for networks and pathways, we prioritized differentially expressed genes with respect to those common to the three TLR ligands as well as those shared by LPS and poly (I:C) but not CpG DNA. The importance of changes in gene expression was demonstrated by experiments indicating that RNA interference-mediated inhibition of two genes identified in this analysis, PLEC1 and TPST1, reduced IL-6 production by J774A.1 and RAW264.7 macrophages stimulated with LPS. Together, these findings delineate macrophage gene response patterns induced by different PAMPs and identify new genes that have not previously been implicated in innate immunity.
PMCID: PMC3163807  PMID: 21665277
innate immunity; gene expression; RNA interference; lipopolysacharide; poly (I:C); CpG DNA; PLEC1; TPST1

Results 1-25 (51)