While earning his M.D. degree in 1991 from Stanford, Dr. Utz co-discovered the transcription factor Nuclear Factor of Activated T Cells (NFAT) with J.P. Shaw in Dr. Gerald Crabtree's laboratory. He completed his internal medicine residency, rheumatology fellowship, and post-doctoral training at Brigham and Women's Hospital in Boston prior to joining the Harvard Medical School Faculty in 1996. He joined the Stanford Faculty in 1999 and was promoted to Associate Professor of Medicine in 2005 and Professor in 2012. Professor Utz is an expert in the study of human and murine autoantibodies and autoantigens, apoptosis signaling pathways, animal models of autoimmunity, proteomics and multiplexed assay development for biomarker discovery.
- Immunology and Rheumatology
Program Director, Medical Science Training Program (MSTP)., Stanford University School of Medicine (2013 - Present)
Program Director, Rheumatology Fellowship Program., Stanford University School of Medicine (2012 - 2013)
MSTP Associate Director, Medical Science Training Program, Stanford University School of Medicine (2009 - 2013)
Member, Committee on Professionalism, Performance, and Promotion, Stanford University School of Medicine (2009 - Present)
Associate Director of Education, Stanford University, ITI Institute (2007 - Present)
Member, Cardiovascular Institute Steering Committee, Stanford University School of Medicine (2007 - 2010)
Treasurer and Secretary, FOCIS, Federal of Clinical Immunology Societies (2007 - 2010)
Board of Directors, Northern California Arthritis Foundation (2008 - 2009)
Appointed Councillor, Clinical Immunology Society (2003 - 2006)
Chair of Education, FOCIS, Federal of Clinical Immunology Societies (2003 - 2007)
Member, MSTP (Medical Science Training Program), Stanford University School of Medicine (2002 - Present)
Member, Dean's Committee on Diversity, Stanford University School of Medicine (2002 - Present)
Member, Immunology Graduate program Predoctoral Committee, Stanford University School of Medicine (2000 - 2003)
Member, Faculty Senate, Stanford Hospital (2000 - 2002)
Member, Internship Selection Committee, Stanford University Hospital (1999 - 2005)
Honors & Awards
Elected Member, American Society for Clinical Investigation (2007)
Elected Member, The Kunkel Society (2006)
Divisional Teaching Award, Stanford University (2002)
Department of Medicine Teaching Award, Stanford University School of Medicine (2008)
Baxter Fellowship, Donald and Delia Baxter (2000)
Faculty Fellows Program, Stanford University School of Medicine (2009)
Immunology Graduate Program Teaching and Mentoring Award, Stanford University (2002)
Proteomics Award Nominee, FOCIS (2000)
Stanford Clinical Medicine Award, Stanford University (1991)
Advanced Predoctoral Fellowship in Pharmacology and Toxicology, Pharmaceutical Mfg. Association (1988-1989)
Merck Student Research Fellowship, American Heart Association (1988-1989)
Delta Epsilon Delta, Stanford University (1988)
Alpha Epsilon Delta, Vice President, Stanford University (1988)
Stanford Medical Scholars Fellowship, Stanford University (1987)
Regina Award for Biology, King's College (1986)
Center for Independent Learning Achievement Award, King's College (1986)
Aquinas Society, Aquinas Society (1985-1986)
Sidney Farber Research Award, Roswell Park Memorial Institute (1985)
CRC Chemistry Award, King's College (1983)
Bausch and Lomb Science Award, Bausch and Lomb (1982)
King's College Scholarship Committee Award, King's College (1982-1986)
King's College Dean's List, summa cum laude, King's College (1982-1986)
Fellowship:Brigham and Women's Hospital Harvard Medical School (1996) MA
Residency:Brigham and Women's Hospital Harvard Medical School (1993) MA
Internship:Brigham and Women's Hospital Harvard Medical School (1992) MA
Board Certification: Rheumatology, American Board of Internal Medicine (1996)
Medical Education:Stanford University School of Medicine (1991) CA
M.D., Stanford University, Medicine (1991)
B.S., King's College, Biology (1986)
Community and International Work
Stanford Institutes of Medicine Summer Research Program, SIMR (formerly CCIS), Stanford University
Training the next generation of scientists
High School students
Opportunities for Student Involvement
Stanford EXPLORE, Stanford Univeristy
A lecture series on Biomedical Research
High School students
Opportunities for Student Involvement
CCIS Summer Student Intern Program, Stanford University
To inspire high school students in research, assist junior faculty with support funding.
Northern California Arthritis Foundation.
community high school students
Opportunities for Student Involvement
Current Research and Scholarly Interests
Members of his laboratory are developing several cutting-edge proteomics technologies for immunological applications, including multiplex planar-based autoantigen microarrays for studying lupus, multiple sclerosis, and other diseases such as diabetes and MCTD; reverse phase protein lysate microarrays for studying blood cells; flow-based assays for analyzing samples from patients; and microfluidic capillary electrophoresis assays. Professor Utz is Founder and Faculty Director of the SIMR Summer High School Research Program, retired Chair of Education for the Federation of Clinical Immunology Societies, and assumed the role of Director of Stanford's Medical Scientist Training Program (MSTP) in August 2013. He provides formal lectures to undergraduate, graduate, and medical students in the Schools of Medicine and Engineering. He also teaches medical students, residents and fellows in the clinics and on the in-patient wards, and has won teaching awards in immunology and in medicine. Professor Utz is a member of the Scientific Advisory Boards of several biotechnology and pharmaceutical companies, and has cofounded 3 Bay Area companies.
The four major goals of our studies are:
(1) To understand the mechanisms by which highly-conserved, diverse molecules and complexes such as histones and splicing particles are targeted by T and B lymphocytes and to determine how an immune response directed against ubiquitous antigens leads to organ-specific autoimmune disease.
(2) To use autoimmune sera as molecular probes to study basic cellular processes, particularly apoptosis signaling pathways, alternative RNA splicing, and endoplasmic reticulum protein transport.
(3) To invent and validate novel technologies for high-throughput, multiplex proteomics signaling molecules and studies. We are currently focusing on proteomics analysis of proteins secreted by immune cells, including cytokines and autoantibodies.
(4) To take advantage of the information provided by autoantibody profiling methods to develop antigen-specific tolerizing therapies for common autoimmune diseases. We collaborate closely with the Stanford laboratory of Dr. Larry Steinman, the principle discoverer of DNA tolerizing technology. Our long-term goal is to develop patient-specific, individualized therapeutics.
- Physician Scientist Hour
INDE 217 (Aut, Win, Spr)
Independent Studies (14)
- Biomedical Informatics Teaching Methods
BIOMEDIN 290 (Aut, Win, Spr, Sum)
- Directed Reading and Research
BIOMEDIN 299 (Aut, Win, Spr, Sum)
- Directed Reading in Immunology
IMMUNOL 299 (Aut, Win, Spr, Sum)
- Directed Reading in Medicine
MED 299 (Aut, Win, Spr, Sum)
- Early Clinical Experience in Immunology
IMMUNOL 280 (Aut, Win, Spr, Sum)
- Early Clinical Experience in Medicine
MED 280 (Aut, Win, Spr, Sum)
- Graduate Research
IMMUNOL 399 (Aut, Win, Spr, Sum)
- Graduate Research
MED 399 (Aut, Win, Spr, Sum)
- Medical Scholars Research
BIOMEDIN 370 (Aut, Win, Spr, Sum)
- Medical Scholars Research
MED 370 (Aut, Win, Spr, Sum)
- Out-of-Department Advanced Research Laboratory in Experimental Biology
BIO 199X (Sum)
- Teaching in Immunology
IMMUNOL 290 (Aut, Win, Spr, Sum)
- Undergraduate Research
IMMUNOL 199 (Aut, Win, Spr, Sum)
- Undergraduate Research
MED 199 (Aut, Win, Spr, Sum)
- Biomedical Informatics Teaching Methods
Prior Year Courses
- Introduction to Writing Research Proposals
INDE 234 (Win)
- MSTP Journal Club
INDE 217 (Win, Spr)
- The Future of Academic Medicine
INDE 200 (Aut)
- The Future of Academic Medicine
INDE 200 (Aut, Win)
- Introduction to Writing Research Proposals
Tetramers reveal IL-17-secreting CD4+ T cells that are specific for U1-70 in lupus and mixed connective tissue disease.
Proceedings of the National Academy of Sciences of the United States of America
2015; 112 (10): 3044-3049
Antigen-specific CD4(+) T cells are implicated in the autoimmune disease systemic lupus erythematosus (SLE), but little is known about the peptide antigens that they recognize and their precise function in disease. We generated a series of MHC class II tetramers of I-E(k)-containing peptides from the spliceosomal protein U1-70 that specifically stain distinct CD4(+) T-cell populations in MRL/lpr mice. The T-cell populations recognize an epitope differing only by the presence or absence of a single phosphate residue at position serine(140). The frequency of CD4(+) T cells specific for U1-70(131-150):I-E(k) (without phosphorylation) correlates with disease severity and anti-U1-70 autoantibody production. These T cells also express RORγt and produce IL-17A. Furthermore, the U1-70-specific CD4(+) T cells that produce IL-17A are detected in a subset of patients with SLE and are significantly increased in patients with mixed connective tissue disease. These studies provide tools for studying antigen-specific CD4(+) T cells in lupus, and demonstrate an antigen-specific source of IL-17A in autoimmune disease.
View details for DOI 10.1073/pnas.1424796112
View details for PubMedID 25713364
Ly108 expression distinguishes subsets of invariant NKT cells that help autoantibody production and secrete IL-21 from those that secrete IL-17 in lupus prone NZB/W mice
JOURNAL OF AUTOIMMUNITY
2014; 50: 87-98
Lupus is a systemic autoimmune disease characterized by anti-nuclear antibodies in humans and genetically susceptible NZB/W mice that can cause immune complex glomerulonephritis. T cells contribute to lupus pathogenesis by secreting pro-inflammatory cytokines such as IL-17, and by interacting with B cells and secreting helper factors such as IL-21 that promote production of IgG autoantibodies. In the current study, we determined whether purified NKT cells or far more numerous conventional non-NKT cells in the spleen of NZB/W female mice secrete IL-17 and/or IL-21 after TCR activation in vitro, and provide help for spontaneous IgG autoantibody production by purified splenic CD19(+) B cells. Whereas invariant NKT cells secreted large amounts of IL-17 and IL-21, and helped B cells, non-NKT cells did not. The subset of IL-17 secreting NZB/W NKT cells expressed the Ly108(lo)CD4(-)NK1.1(-) phenotype, whereas the IL-21 secreting subset expressed the Ly108(hi)CD4(+)NK1.1(-) phenotype and helped B cells secrete a variety of IgG anti-nuclear antibodies. α-galactocylceramide enhanced the helper activity of NZB/W and B6.Sle1b NKT cells for IgG autoantibody secretion by syngeneic B cells. In conclusion, different subsets of iNKT cells from mice with genetic susceptibility to lupus can contribute to pathogenesis by secreting pro-inflammatory cytokines and helping autoantibody production.
View details for DOI 10.1016/j.jaut.2014.01.002
View details for Web of Science ID 000336114600012
Protein microarray analysis reveals BAFF-binding autoantibodies in systemic lupus erythematosus
JOURNAL OF CLINICAL INVESTIGATION
2013; 123 (12): 5135-5145
Autoantibodies against cytokines, chemokines, and growth factors inhibit normal immunity and are implicated in inflammatory autoimmune disease and diseases of immune deficiency. In an effort to evaluate serum from autoimmune and immunodeficient patients for Abs against cytokines, chemokines, and growth factors in a high-throughput and unbiased manner, we constructed a multiplex protein microarray for detection of serum factor-binding Abs and used the microarray to detect autoantibody targets in SLE. We designed a nitrocellulose-surface microarray containing human cytokines, chemokines, and other circulating proteins and demonstrated that the array permitted specific detection of serum factor-binding probes. We used the arrays to detect previously described autoantibodies against cytokines in samples from individuals with autoimmune polyendocrine syndrome type 1 and chronic mycobacterial infection. Serum profiling from individuals with SLE revealed that among several targets, elevated IgG autoantibody reactivity to B cell-activating factor (BAFF) was associated with SLE compared with control samples. BAFF reactivity correlated with the severity of disease-associated features, including IFN-α-driven SLE pathology. Our results showed that serum factor protein microarrays facilitate detection of autoantibody reactivity to serum factors in human samples and that BAFF-reactive autoantibodies may be associated with an elevated inflammatory disease state within the spectrum of SLE.
View details for DOI 10.1172/JCI70231
View details for Web of Science ID 000327826100020
View details for PubMedID 24270423
Clinical optimization of antigen specific modulation of type 1 diabetes with the plasmid DNA platform
2013; 149 (3): 297-306
Some clinical trials in humans have aimed at modulation of type 1 diabetes (T1D) via alteration of the immune response to putative islet cell antigens, particularly proinsulin and insulin, glutamic acid decarboxylase and the peptide, DiaPep 277, derived from heat shock protein 60. The focus here is on development of a specially engineered DNA plasmid encoding proinsulin to treat T1D. The plasmid is engineered to turn off adaptive immunity to proinsulin. This approach yielded exciting results in a randomized placebo controlled trial in 80 adult patients with T1D. The implications of this trial are explored in regards to the potential for sparing inflammation in islets and thus allowing the functioning beta cells to recover and produce more insulin. Strategies to further strengthen the effects seen thus far with the tolerizing DNA plasmid to proinsulin will be elucidated. The DNA platform affords an opportunity for easy modifications. In addition standard exploration of dose levels, route of administration and frequency of dose are practical. Optimization of the effects seen to date on C-peptide and on depletion of proinsulin specific CD8 T cells are feasible, with expected concomitant improvement in other parameters like hemoglobin A1c and reduction in insulin usage. T1D is one of the few autoimmune conditions where antigen specific therapy can be achieved, provided the approach is tested intelligently. Tolerizing DNA vaccines to proinsulin and other islet cell autoantigens is a worthy pursuit to potentially treat, prevent and to perhaps even 'cure' or 'prevent' type 1 diabetes.
View details for DOI 10.1016/j.clim.2013.08.010
View details for Web of Science ID 000328874700005
View details for PubMedID 24094739
- Brief Report: Interferon- Induction and Detection of Anti-Ro, Anti-La, Anti-Sm, and Anti-RNP Autoantibodies by Autoantigen Microarray Analysis in Juvenile Dermatomyositis ARTHRITIS AND RHEUMATISM 2013; 65 (9): 2424-2429
- An Integrated Peptide-Antigen Microarray on Plasmonic Gold Films for Sensitive Human Antibody Profiling PLOS ONE 2013; 8 (7)
Plasmid-Encoded Proinsulin Preserves C-Peptide While Specifically Reducing Proinsulin-Specific CD8+ T Cells in Type 1 Diabetes.
Science translational medicine
2013; 5 (191): 191ra82-?
In type 1 diabetes (T1D), there is an intense inflammatory response that destroys the ? cells in the pancreatic islets of Langerhans, the site where insulin is produced and released. A therapy for T1D that targets the specific autoimmune response in this disease while leaving the remainder of the immune system intact, has long been sought. Proinsulin is a major target of the adaptive immune response in T1D. We hypothesized that an engineered DNA plasmid encoding proinsulin (BHT-3021) would preserve ? cell function in T1D patients through reduction of insulin-specific CD8(+) T cells. We studied 80 subjects over 18 years of age who were diagnosed with T1D within the past 5 years. Subjects were randomized 2:1 to receive intramuscular injections of BHT-3021 or BHT-placebo, weekly for 12 weeks, and then monitored for safety and immune responses in a blinded fashion. Four dose levels of BHT-3021 were evaluated: 0.3, 1.0, 3.0, and 6.0 mg. C-peptide was used both as an exploratory efficacy measure and as a safety measure. Islet-specific CD8(+) T cell frequencies were assessed with multimers of monomeric human leukocyte antigen class I molecules loaded with peptides from pancreatic and unrelated antigens. No serious adverse events related to BHT-3021 were observed. C-peptide levels improved relative to placebo at all doses, at 1 mg at the 15-week time point (+19.5% BHT-3021 versus -8.8% BHT-placebo, P < 0.026). Proinsulin-reactive CD8(+) T cells, but not T cells against unrelated islet or foreign molecules, declined in the BHT-3021 arm (P < 0.006). No significant changes were noted in interferon-?, interleukin-4 (IL-4), or IL-10 production in CD4 T cells. Thus, we demonstrate that a plasmid encoding proinsulin reduces the frequency of CD8(+) T cells reactive to proinsulin while preserving C-peptide over the course of dosing.
View details for DOI 10.1126/scitranslmed.3006103
View details for PubMedID 23803704
- Characterization of Influenza Vaccine Immunogenicity Using Influenza Antigen Microarrays PLOS ONE 2013; 8 (5)
- Apoptosis and other immune biomarkers predict influenza vaccine responsiveness MOLECULAR SYSTEMS BIOLOGY 2013; 9
Granzyme B and natural killer (NK) cell death.
2005; 15 (5): 315-322
Granzyme B is a unique serine protease, which plays a crucial role for target cell death. Several mechanisms of delivery of granzyme B to target cells have been recently identified. Granzyme B directly activates Bid, a specific substrate for granzyme B, resulting in caspase activation. Granzyme B efficiently cleaves many prominent autoantigens, and the hypothesis that autoantibodies arise when cryptic determinants are revealed to the immune system has been proposed. Some autoantibodies directed against granzyme B-specific neoepitopes are present in serum from patients with autoimmune diseases. In the tissues from autoimmune diseases, granzyme B might play an important role for disease progression (i.e., rheumatoid arthritis synovium) or inhibition (i.e., regulatory T cells). We have identified a novel type of activation-induced cell death (granzyme B leakage-induced cell death). Activation-induced natural killer (NK) cell death is accompanied by the leakage of granzyme B from intracellular granules into the cytoplasm, and it triggers apoptosis by directing Bid to mitochondrial membranes. An excess of "leaked" granzyme B over its inhibitor, serpin proteinase inhibitor 9, is a major determinant of cell death. The role of granzyme B in autoimmunity and its influence on NK cell death are discussed.
View details for PubMedID 17029086
Small nucleolar RNP scleroderma autoantigens associate with phosphorylated serine/arginine splicing factors during apoptosis
ARTHRITIS AND RHEUMATISM
2000; 43 (6): 1327-1336
Proteins that are phosphorylated during apoptosis are commonly precipitated by autoantibodies found in the sera of patients with systemic lupus erythematosus. We sought to determine whether scleroderma autoantigens such as small nucleolar RNPs (snoRNP) also associate with phosphoproteins in response to various cellular stressors.We screened a panel of monoclonal antibodies derived from mice exposed to mercury, a well-characterized murine model of the anti-snoRNP autoimmune response, for the ability to selectively precipitate phosphoproteins from radiolabeled lysates prepared from Jurkat T cells subjected to stressful stimuli.Monoclonal antibodies reactive with snoRNPs precipitated a phosphoprotein complex (pp42, pp34, and pp23) from lysates prepared from apoptotic cells. Several novel phosphoproteins (pp62 and pp18) were also observed. The phosphorylation and/or recruitment of these proteins to the snoRNP complex is induced by multiple apoptotic stimuli (e.g., Fas ligation, anisomycin, or ultraviolet irradiation), an effect that is blocked by overexpression of Bcl-2. We were unable to demonstrate an association of the phosphoprotein complex with snoRNPs in cells treated with the xenobiotic agent mercury. The snoRNP-associated phosphoprotein complex is composed of serine/arginine (SR) splicing factors, including SRp40.The association of phosphorylated SR proteins with snoRNPs in cells undergoing apoptosis suggests that the immune response to fibrillarin that characterizes a subset of patients with scleroderma may be related to cell death induced by apoptotic stimuli (e.g., Fas ligation, irradiation, or chemical toxins), or by exposure to mercury.
View details for Web of Science ID 000087503300015
View details for PubMedID 10857791
Broad-Scale Phosphoprotein Profiling of Beta Adrenergic Receptor (beta-AR) Signaling Reveals Novel Phosphorylation and Dephosphorylation Events
2013; 8 (12)
β-adrenergic receptors (β-ARs) are model G-protein coupled receptors that mediate signal transduction in the sympathetic nervous system. Despite the widespread clinical use of agents that target β-ARs, the signaling pathways that operate downstream of β-AR stimulation have not yet been completely elucidated. Here, we utilized a lysate microarray approach to obtain a broad-scale perspective of phosphoprotein signaling downstream of β-AR. We monitored the time course of phosphorylation states of 54 proteins after β-AR activation mouse embryonic fibroblast (MEF) cells. In response to stimulation with the non-selective β-AR agonist isoproterenol, we observed previously described phosphorylation events such as ERK1/2(T202/Y204) and CREB(S133), but also novel phosphorylation events such as Cdc2(Y15) and Pyk2(Y402). All of these events were mediated through cAMP and PKA as they were reproduced by stimulation with the adenylyl cyclase activator forskolin and were blocked by treatment with H89, a PKA inhibitor. In addition, we also observed a number of novel isoproterenol-induced protein dephosphorylation events in target substrates of the PI3K/AKT pathway: GSK3β(S9), 4E-BP1(S65), and p70s6k(T389). These dephosphorylations were dependent on cAMP, but were independent of PKA and correlated with reduced PI3K/AKT activity. Isoproterenol stimulation also led to a cAMP-dependent dephosphorylation of PP1α(T320), a modification known to correlate with enhanced activity of this phosphatase. Dephosphorylation of PP1α coincided with the secondary decline in phosphorylation of some PKA-phosphorylated substrates, suggesting that PP1α may act in a feedback loop to return these phosphorylations to baseline. In summary, lysate microarrays are a powerful tool to profile phosphoprotein signaling and have provided a broad-scale perspective of how β-AR signaling can regulate key pathways involved in cell growth and metabolism.
View details for DOI 10.1371/journal.pone.0082164
View details for Web of Science ID 000328566100089
View details for PubMedID 24340001
Interferon-a induction and detection of anti-ro, anti-la, anti-sm, and anti-rnp autoantibodies by autoantigen microarray analysis in juvenile dermatomyositis.
Arthritis and rheumatism
2013; 65 (9): 2424-2429
Objective. To evaluate serum interferon-alpha (interferon-α) activity in the context of autoantibody profiles in juvenile dermatomyositis (JDM) patients. Methods. Sera from 36 JDM patients were analyzed. Autoantibody profiles were determined by probing microarrays, fabricated with ~80 distinct autoantigens, with serum and a Cy3-conjugated secondary antibody. Arrays were scanned and analyzed to determine antigen reactivity. Serum interferon-α activity was measured using a functional reporter cell assay. Sera were assayed alone or in combination with cellular material released from necrotic U937 cells to stimulate peripheral blood mononuclear cells in vitro from healthy donors, and interferon-α production in culture was measured by a dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA). Results. 75% of JDM sera reacted to at least one of forty-one autoantigens on the microarray, including Ro52, Ro60, La, Smith and ribonucleoprotein (RNP). Seven samples were positive in the reporter cell assay. There was a significant association between reactivity against Ro, La, Smith and proliferating cell nuclear antigen and serum interferon-α activity in this assay (p=0.005). Significance analysis of microarrays (SAM) identified increased reactivity against Smith, RNP, Ro52, U1-C and Mi-2 in these sera. Sixteen samples induced interferon-α production as measured by DELFIA. There was a significant association between reactivity against Ro, La, Smith and RNP and the induction of interferon-α with serum and necrotic cell material (p=0.035). SAM identified increased reactivity against Ro60 in these sera. Conclusion. These data support the hypothesis that nucleic-acid associated autoantibodies, including the Ro/La and Smith/RNP complexes, may stimulate the production of active interferon- α in children with JDM. © 2013 American College of Rheumatology.
View details for DOI 10.1002/art.38038
View details for PubMedID 23740815
- Multiplexed cytokine detection on plasmonic gold substrates with enhanced near-infrared fluorescence NANO RESEARCH 2013; 6 (2): 113-120
Characterization of influenza vaccine immunogenicity using influenza antigen microarrays.
2013; 8 (5)
Existing methods to measure influenza vaccine immunogenicity prohibit detailed analysis of epitope determinants recognized by immunoglobulins. The development of highly multiplex proteomics platforms capable of capturing a high level of antibody binding information will enable researchers and clinicians to generate rapid and meaningful readouts of influenza-specific antibody reactivity.We developed influenza hemagglutinin (HA) whole-protein and peptide microarrays and validated that the arrays allow detection of specific antibody reactivity across a broad dynamic range using commercially available antibodies targeted to linear and conformational HA epitopes. We derived serum from blood draws taken from 76 young and elderly subjects immediately before and 28±7 days post-vaccination with the 2008/2009 trivalent influenza vaccine and determined the antibody reactivity of these sera to influenza array antigens.Using linear regression and correcting for multiple hypothesis testing by the Benjamini and Hochberg method of permutations over 1000 resamplings, we identified antibody reactivity to influenza whole-protein and peptide array features that correlated significantly with age, H1N1, and B-strain post-vaccine titer as assessed through a standard microneutralization assay (p<0.05, q <0.2). Notably, we identified several peptide epitopes that were inversely correlated with regard to age and seasonal H1N1 and B-strain neutralization titer (p<0.05, q <0.2), implicating reactivity to these epitopes in age-related defects in response to H1N1 influenza. We also employed multivariate linear regression with cross-validation to build models based on age and pre-vaccine peptide reactivity that predicted vaccine-induced neutralization of seasonal H1N1 and H3N2 influenza strains with a high level of accuracy (84.7% and 74.0%, respectively).Our methods provide powerful tools for rapid and accurate measurement of broad antibody-based immune responses to influenza, and may be useful in measuring response to other vaccines and infectious agents.
View details for DOI 10.1371/journal.pone.0064555
View details for PubMedID 23734205
Apoptosis and other immune biomarkers predict influenza vaccine responsiveness.
Molecular systems biology
2013; 9: 659-?
Despite the importance of the immune system in many diseases, there are currently no objective benchmarks of immunological health. In an effort to identifying such markers, we used influenza vaccination in 30 young (20-30 years) and 59 older subjects (60 to >89 years) as models for strong and weak immune responses, respectively, and assayed their serological responses to influenza strains as well as a wide variety of other parameters, including gene expression, antibodies to hemagglutinin peptides, serum cytokines, cell subset phenotypes and in vitro cytokine stimulation. Using machine learning, we identified nine variables that predict the antibody response with 84% accuracy. Two of these variables are involved in apoptosis, which positively associated with the response to vaccination and was confirmed to be a contributor to vaccine responsiveness in mice. The identification of these biomarkers provides new insights into what immune features may be most important for immune health.
View details for DOI 10.1038/msb.2013.15
View details for PubMedID 23591775
Redirecting cell-type specific cytokine responses with engineered interleukin-4 superkines
NATURE CHEMICAL BIOLOGY
2012; 8 (12): 990-998
Cytokines dimerize their receptors, with the binding of the 'second chain' triggering signaling. In the interleukin (IL)-4 and IL-13 system, different cell types express varying numbers of alternative second receptor chains (?c or IL-13R?1), forming functionally distinct type I or type II complexes. We manipulated the affinity and specificity of second chain recruitment by human IL-4. A type I receptor-selective IL-4 'superkine' with 3,700-fold higher affinity for ?c was three- to ten-fold more potent than wild-type IL-4. Conversely, a variant with high affinity for IL-13R?1 more potently activated cells expressing the type II receptor and induced differentiation of dendritic cells from monocytes, implicating the type II receptor in this process. Superkines showed signaling advantages on cells with lower second chain numbers. Comparative transcriptional analysis reveals that the superkines induce largely redundant gene expression profiles. Variable second chain numbers can be exploited to redirect cytokines toward distinct cell subsets and elicit new actions, potentially improving the selectivity of cytokine therapy.
View details for DOI 10.1038/NCHEMBIO.1096
View details for Web of Science ID 000311491200014
View details for PubMedID 23103943
Using the allergic immune system to target cancer: activity of IgE antibodies specific for human CD20 and MUC1
CANCER IMMUNOLOGY IMMUNOTHERAPY
2012; 61 (12): 2295-2309
Monoclonal antibodies are widely used in the treatment of many B cell lymphomas and certain solid tumors. All currently approved therapeutic monoclonal antibodies are of the immunoglobulin G (IgG) isotype. We hypothesized that tumor-specific monoclonal antibodies of the IgE isotype may serve as effective cancer therapeutics. To test this hypothesis, we produced mouse-human chimeric IgE antibodies specific for the human B cell antigen CD20 and the epithelial antigen MUC1. We demonstrate here that anti-hCD20 IgE antibodies have in vitro cytotoxic activity when used with purified allergic effector cells derived from umbilical cord blood. At an effector-tumor ratio of 2:1, mast cells and tumor-specific IgE induced a 2.5-fold increase in tumor cell death, as compared to control IgE. Similar results were observed when eosinophils were used as effector cells. In an in vivo murine model of breast carcinoma, administration of anti-hMUC1 IgE reduced the growth of MUC1(+) tumors by 25-30 % in hFc?RI transgenic mice. In contrast, local production of IgE and cytokines chemotactic for macrophages, eosinophils and mast cells led to complete tumor eradication. These results suggest that allergic effector cells activated by IgE and cell surface antigens have the capacity to induce tumor cell death in vitro and in vivo. The use of chimeric antibodies and hFc?RI transgenic mice will greatly enhance investigations in the nascent field of allergo-oncology.
View details for DOI 10.1007/s00262-012-1299-0
View details for Web of Science ID 000311666600009
View details for PubMedID 22692757
On silico peptide microarrays for high-resolution mapping of antibody epitopes and diverse protein-protein interactions
2012; 18 (9): 1434-?
We developed a new, silicon-based peptide array for a broad range of biological applications, including potential development as a real-time point-of-care platform. We used photolithography on silicon wafers to synthesize microarrays (Intel arrays) that contained every possible overlapping peptide within a linear protein sequence covering the N-terminal tail of human histone H2B. These arrays also included peptides with acetylated and methylated lysine residues, reflecting post-translational modifications of H2B. We defined minimum binding epitopes for commercial antibodies recognizing the modified and unmodified H2B peptides. We further found that this platform is suitable for the highly sensitive characterization of methyltransferases and kinase substrates. The Intel arrays also revealed specific H2B epitopes that are recognized by autoantibodies in individuals with systemic lupus erythematosus who have elevated disease severity. By combining emerging nonfluorescence-based detection methods with an underlying integrated circuit, we are now poised to create a truly transformative proteomics platform with applications in bioscience, drug development and clinical diagnostics.
View details for DOI 10.1038/nm.2913
View details for Web of Science ID 000308472300042
View details for PubMedID 22902875
New tools for classification and monitoring of autoimmune diseases
NATURE REVIEWS RHEUMATOLOGY
2012; 8 (6): 317-328
Rheumatologists see patients with a range of autoimmune diseases. Phenotyping these diseases for diagnosis, prognosis and selection of therapies is an ever increasing problem. Advances in multiplexed assay technology at the gene, protein, and cellular level have enabled the identification of 'actionable biomarkers'; that is, biological metrics that can inform clinical practice. Not only will such biomarkers yield insight into the development, remission, and exacerbation of a disease, they will undoubtedly improve diagnostic sensitivity and accuracy of classification, and ultimately guide treatment. This Review provides an introduction to these powerful technologies that could promote the identification of actionable biomarkers, including mass cytometry, protein arrays, and immunoglobulin and T-cell receptor high-throughput sequencing. In our opinion, these technologies should become part of routine clinical practice for the management of autoimmune diseases. The use of analytical tools to deconvolve the data obtained from use of these technologies is also presented here. These analyses are revealing a more comprehensive and interconnected view of the immune system than ever before and should have an important role in directing future treatment approaches for autoimmune diseases.
View details for DOI 10.1038/nrrheum.2012.66
View details for Web of Science ID 000304719600005
View details for PubMedID 22647780
Differential mTOR and ERK pathway utilization by effector CD4 T cells suggests combinatorial drug therapy of arthritis
2012; 142 (2): 127-138
The signaling pathways utilized by naïve and experienced effector CD4 T cells during activation and proliferation were evaluated. While inhibition of either mTOR or MAPK alone was able to inhibit naïve T cell proliferation, both mTOR and MAPK (ERK) pathway inhibition was required to efficiently block experienced, effector CD4 T cell proliferation. This was demonstrated both in vitro, and in vivo by treating mice with collagen-induced arthritis using mTOR and/or ERK inhibitors. The combination of mTOR and ERK inhibition prevented or treated disease more efficiently than either agent alone. These data illustrate the different requirements of naïve and experienced effector CD4 T cells in the use of the mTOR and MAPK pathways in proliferation, and suggest that therapies targeting both the mTOR and MAPK pathways may be more effective than targeting either pathway alone in the treatment of CD4 T cell-mediated autoimmunity.
View details for DOI 10.1016/j.clim.2011.09.008
View details for Web of Science ID 000300388200007
View details for PubMedID 22075384
- Therapeutic Toll-like receptor agonists directly influence mouse and human T cell lymphoma cell viability and cytokine secretion LEUKEMIA & LYMPHOMA 2012; 53 (1): 166-168
Specific post-translational histone modifications of neutrophil extracellular traps as immunogens and potential targets of lupus autoantibodies
ARTHRITIS RESEARCH & THERAPY
2012; 14 (1)
Autoreactivity to histones is a pervasive feature of several human autoimmune disorders, including systemic lupus erythematosus (SLE). Specific post-translational modifications (PTMs) of histones within neutrophil extracellular traps (NETs) may potentially drive the process by which tolerance to these chromatin-associated proteins is broken. We hypothesized that NETs and their unique histone PTMs might be capable of inducing autoantibodies that target histones.We developed a novel and efficient method for the in vitro production, visualization, and broad profiling of histone-PTMs of human and murine NETs. We also immunized Balb/c mice with murine NETs and profiled their sera on autoantigen and histone peptide microarrays for evidence of autoantibody production to their immunogen.We confirmed specificity toward acetyl-modified histone H2B as well as to other histone PTMs in sera from patients with SLE known to have autoreactivity against histones. We observed enrichment for distinctive histone marks of transcriptionally silent DNA during NETosis triggered by diverse stimuli. However, NETs derived from human and murine sources did not harbor many of the PTMs toward which autoreactivity was observed in patients with SLE or in MRL/lpr mice. Further, while murine NETs were weak autoantigens in vivo, there was only partial overlap in the immunoglobulin G (IgG) and IgM autoantibody profiles induced by vaccination of mice with NETs and those seen in patients with SLE.Isolated in vivo exposure to NETs is insufficient to break tolerance and may involve additional factors that have yet to be identified.
View details for DOI 10.1186/ar3707
View details for Web of Science ID 000304698800039
View details for PubMedID 22300536
- Correction: Specific post-translational histone modifications of neutrophil extracellular traps as immunogens and potential targets of lupus autoantibodies. Arthritis research & therapy 2012; 14 (4): 403-?
A proteomic approach for the identification of novel lysine methyltransferase substrates
EPIGENETICS & CHROMATIN
Signaling via protein lysine methylation has been proposed to play a central role in the regulation of many physiologic and pathologic programs. In contrast to other post-translational modifications such as phosphorylation, proteome-wide approaches to investigate lysine methylation networks do not exist.In the current study, we used the ProtoArray® platform, containing over 9,500 human proteins, and developed and optimized a system for proteome-wide identification of novel methylation events catalyzed by the protein lysine methyltransferase (PKMT) SETD6. This enzyme had previously been shown to methylate the transcription factor RelA, but it was not known whether SETD6 had other substrates. By using two independent detection approaches, we identified novel candidate substrates for SETD6, and verified that all targets tested in vitro and in cells were genuine substrates.We describe a novel proteome-wide methodology for the identification of new PKMT substrates. This technological advance may lead to a better understanding of the enzymatic activity and substrate specificity of the large number (more than 50) PKMTs present in the human proteome, most of which are uncharacterized.
View details for DOI 10.1186/1756-8935-4-19
View details for Web of Science ID 000296832600001
View details for PubMedID 22024134
T(H)1, T(H)2, and T(H)17 cells instruct monocytes to differentiate into specialized dendritic cell subsets
2011; 118 (12): 3311-3320
Monocytes and T helper (T(H)) cells rapidly infiltrate inflamed tissues where monocytes differentiate into inflammatory dendritic cells (DCs) through undefined mechanisms. Our studies indicate that T(H) cells frequently interact with monocytes in inflamed skin and elicit the differentiation of specialized DC subsets characteristic of these lesions. In psoriasis lesions, T(H)1 and T(H)17 cells interact with monocytes and instruct these cells to differentiate into T(H)1- and T(H)17-promoting DCs, respectively. Correspondingly, in acute atopic dermatitis, T(H)2 cells interact with monocytes and elicit the formation of T(H)2-promoting DCs. DC formation requires GM-CSF and cell contact, whereas T(H) subset specific cytokines dictate DC function and the expression of DC subset specific surface molecules. Moreover, the phenotypes of T cell-induced DC subsets are maintained after subsequent stimulation with a panel of TLR agonists, suggesting that T(H)-derived signals outweigh downstream TLR signals in their influence on DC function. These findings indicate that T(H) cells govern the formation and function of specialized DC subsets.
View details for DOI 10.1182/blood-2011-03-341065
View details for Web of Science ID 000295120900018
View details for PubMedID 21813450
CpG and Non-CpG Oligodeoxynucleotides Directly Costimulate Mouse and Human CD4(+) T Cells through a TLR9-and MyD88-Independent Mechanism
JOURNAL OF IMMUNOLOGY
2011; 187 (6): 3033-3043
TLR ligands are known to activate APCs, but direct T cell responsiveness to TLR ligands is controversial. Because of their clinical relevance, we performed in-depth studies of the effects of the TLR9-associated ligands, oligodeoxynucleotides (ODNs), on highly purified T lymphocytes. Both CpG and non-CpG ODNs directly costimulate mouse and human CD4(+) T cells, resulting in activation marker upregulation, cytokine secretion, elevated TCR phosphorylation, and proliferation. Surprisingly, ODN costimulation occurred independently of TLR9 and MyD88, as well as ICOS, CD28, and TRIF. TLR9-antagonist ODNs likewise promoted T cell activation, which has important implications for the study of these "inhibitory" ODNs in inflammatory diseases. Cytokine profiling revealed that ODNs promote polarization of distinct Th subsets, and that ODNs differentially affect human naive and memory T cells. Our studies reveal a striking and unexpected ability of ODNs to directly activate and polarize T cells, presenting an opportunity to enhance the paradigm for selection of therapeutic ODNs in humans.
View details for DOI 10.4049/jimmunol.1003414
View details for Web of Science ID 000295034200022
View details for PubMedID 21844387
Plasmonic substrates for multiplexed protein microarrays with femtomolar sensitivity and broad dynamic range
Protein chips are widely used for high-throughput proteomic analysis, but to date, the low sensitivity and narrow dynamic range have limited their capabilities in diagnostics and proteomics. Here we present protein microarrays on a novel nanostructured, plasmonic gold film with near-infrared fluorescence enhancement of up to 100-fold, extending the dynamic range of protein detection by three orders of magnitude towards the fM regime. We employ plasmonic protein microarrays for the early detection of a cancer biomarker, carcinoembryonic antigen, in the sera of mice bearing a xenograft tumour model. Further, we demonstrate a multiplexed autoantigen array for human autoantibodies implicated in a range of autoimmune diseases with superior signal-to-noise ratios and broader dynamic range compared with commercial nitrocellulose and glass substrates. The high sensitivity, broad dynamic range and easy adaptability of plasmonic protein chips presents new opportunities in proteomic research and diagnostics applications.
View details for DOI 10.1038/ncomms1477
View details for Web of Science ID 000294807200008
View details for PubMedID 21915108
- SCREENING CyTOF-the next generation of cell detection NATURE REVIEWS RHEUMATOLOGY 2011; 7 (9): 502-503
Reactivity profiles of broadly neutralizing anti-HIV-1 antibodies are distinct from those of pathogenic autoantibodies
2011; 25 (10): 1247-1257
Broadly neutralizing antibodies (bNt Abs) against HIV-1 are rarely produced during natural infection, and efforts to induce such Abs by vaccination have been unsuccessful. Thus, elucidating the nature and cellular origins of bNt Abs is a high priority for vaccine research. As the bNt monoclonal Abs (MAbs) 2F5, 4E10 and 2G12 have been reported to bind select autoantigens, we investigated whether these MAbs display a broader range of autoreactivity and how their autoreactivity compares with that of pathogenic autoAbs.An autoantigen microarray comprising 106 connective tissue disease-related autoantigens and control antigens was developed and used, in combination with ELISAs, to compare the reactivity profiles of MAbs 4E10, 2F5 and 2G12 to those of four pathogenic autoAbs derived from patients with antiphospholipid-syndrome (APS), and to serum from a patient with systemic lupus erythematosus (SLE).The APS MAbs and SLE serum reacted strongly with multiple autoantigens on the microarray, whereas anti-HIV-1 MAb reactivity was limited mainly to HIV-1-related antigens. The APS autoAbs reacted strongly with CL, yet only 4E10 bound CL at high concentrations; both 2F5 and 4E10 bound their HIV-1 epitopes with a 2-3-log higher apparent affinity than CL. Moreover, the polyreactivity of 4E10, but not CL15, could be blocked with dried milk.The reactivity profiles of bNt anti-HIV-1 MAbs are fundamentally distinct from those of pathogenic autoAbs that arise from dysregulated tolerance mechanisms. This suggests that the limited polyreactivity observed for the bNt MAbs, and for HIV-1-Nt Abs in general, may arise through alternative mechanisms, such as extensive somatic mutation due to persistent antigen selection during chronic infection.
View details for DOI 10.1097/QAD.0b013e32834785cf
View details for Web of Science ID 000291463200001
View details for PubMedID 21508803
Lysine methylation of the NF-kappa B subunit RelA by SETD6 couples activity of the histone methyltransferase GLP at chromatin to tonic repression of NF-kappa B signaling
2011; 12 (1): 29-U47
Signaling via the methylation of lysine residues in proteins has been linked to diverse biological and disease processes, yet the catalytic activity and substrate specificity of many human protein lysine methyltransferases (PKMTs) are unknown. We screened over 40 candidate PKMTs and identified SETD6 as a methyltransferase that monomethylated chromatin-associated transcription factor NF-?B subunit RelA at Lys310 (RelAK310me1). SETD6-mediated methylation rendered RelA inert and attenuated RelA-driven transcriptional programs, including inflammatory responses in primary immune cells. RelAK310me1 was recognized by the ankryin repeat of the histone methyltransferase GLP, which under basal conditions promoted a repressed chromatin state at RelA target genes through GLP-mediated methylation of histone H3 Lys9 (H3K9). NF-?B-activation-linked phosphorylation of RelA at Ser311 by protein kinase C-? (PKC-?) blocked the binding of GLP to RelAK310me1 and relieved repression of the target gene. Our findings establish a previously uncharacterized mechanism by which chromatin signaling regulates inflammation programs.
View details for DOI 10.1038/ni.1968
View details for Web of Science ID 000285465100010
View details for PubMedID 21131967
Regulation of human Th9 differentiation by type I interferons and IL-21
IMMUNOLOGY AND CELL BIOLOGY
2010; 88 (6): 624-631
Interleukin (IL)-9-producing CD4(+) T cells are a novel subset of T helper (Th) cells that develops independently of the Th1, Th2, Th17 and regulatory T-cell lineages. Similar to the murine model, transforming growth factor (TGF)-beta and IL-4 directed human naive CD4(+) T cells to produce IL-9. Whereas IL-4 suppressed TGF-beta-induced Foxp3 expression, TGF-beta failed to inhibit IL-4-mediated upregulation of the Th2 transcription factor GATA-3. Addition of IL-1 beta, IL-6, IL-10, interferon (IFN)-alpha, IFN-beta or IL-21 to Th9-polarizing conditions augmented Th9 differentiation, while the Th1-associated cytokines IFN-gamma and IL-27 partially suppressed IL-9 production. Given that T cells are a primary source of IL-21, IL-21 expression was analyzed under Th9-polarizing conditions in the context of inflammatory cytokines. Surprisingly, type I IFNs induced elevated levels of IL-21, and blockade of IL-21 abrogated their ability to enhance Th9 differentiation. Taken together, these data indicate a complex cytokine network in the regulation of human IL-9-producing CD4(+) T cells.
View details for DOI 10.1038/icb.2010.53
View details for Web of Science ID 000280830700007
View details for PubMedID 20421880
Treatment with a Toll-like receptor inhibitory GpG oligonucleotide delays and attenuates lupus nephritis in NZB/W mice
2010; 43 (2): 140-155
Activation of the innate immune system by DNA containing hypomethylated CpG motifs has been implicated in the pathogenesis of systemic lupus erythematosus (SLE). Here, we examined the consequences of immunostimulatory CpG-oligodeoxynucleotide (ODN) and inhibitory GpG-ODN treatment in the NZB x NZW F1 (NZB/W) murine model of SLE. Beginning at 5 months of age, we administered CpG-ODN or GpG-ODN at regular intervals to female NZB/W animals. We also determined the effects of ODN administration on NZB/W mouse lymphocyte function, and the specificity of ODN binding to Toll-like receptors (TLRs) other than TLR-9. While CpG-ODN treatment did not appear to have a major impact on disease severity, GpG-ODN treatment significantly delayed the onset of proteinuria in NZB/W mice. Interestingly, short-term GpG-ODN treatment promoted Th2-type T and B cell responses, and inhibited B lymphocyte proliferation in vitro. On the other hand, extended GpG-ODN treatment did not result in sustained Th2 responses or significantly reduced renal disease. Moreover, the binding of CpG-ODN and GpG-ODN was not restricted to TLR-9 as both ODNs also interacted with TLR-3, TLR-7, and TLR-8. Taken together, the data indicate that the protective mechanism of GpG-ODN treatment in the NZB/W model of lupus nephritis involves modulating T cell cytokine profiles and B lymphocyte activation through the inhibition of several TLRs, including TLR-7 and TLR-9.
View details for DOI 10.3109/08916930903229239
View details for Web of Science ID 000275059900004
View details for PubMedID 19845477
The U1-snRNP complex: structural properties relating to autoimmune pathogenesis in rheumatic diseases
2010; 233: 126-145
The U1 small nuclear ribonucleoprotein particle (snRNP) is a target of autoreactive B cells and T cells in several rheumatic diseases including systemic lupus erythematosus (SLE) and mixed connective tissue disease (MCTD). We propose that inherent structural properties of this autoantigen complex, including common RNA-binding motifs, B and T-cell epitopes, and a unique stimulatory RNA molecule, underlie its susceptibility as a target of the autoimmune response. Immune mechanisms that may contribute to overall U1-snRNP immunogenicity include epitope spreading through B and T-cell interactions, apoptosis-induced modifications, and toll-like receptor (TLR) activation through stimulation by U1-snRNA. We conclude that understanding the interactions between U1-snRNP and the immune system will provide insights into why certain patients develop anti-U1-snRNP autoimmunity, and more importantly how to effectively target therapies against this autoimmune response.
View details for Web of Science ID 000273067700009
View details for PubMedID 20192997
- Targeting mTOR and MAPK Pathways to Inhibit Naive and Experienced Effector CD4 T Cells in Autoimmunity ACADEMIC PRESS INC ELSEVIER SCIENCE. 2010: S69-S69
Cholinergic Modulation of Angiogenesis: Role of the 7 Nicotinic Acetylcholine Receptor
JOURNAL OF CELLULAR BIOCHEMISTRY
2009; 108 (2): 433-446
Pathological angiogenesis contributes to tobacco-related diseases such as malignancy, atherosclerosis and age-related macular degeneration. Nicotine acts on endothelial nicotinic acetylcholine receptors (nAChRs) to activate endothelial cells and to augment pathological angiogenesis. In the current study, we studied nAChR subunits involved in these actions. We detected mRNA for all mammalian nAChR subunits except alpha(2), alpha(4), gamma, and delta in four different types of ECs. Using siRNA methodology, we found that the alpha(7) nAChR plays a dominant role in nicotine-induced cell signaling (assessed by intracellular calcium and NO imaging, and studies of protein expression and phosphorylation), as well as nicotine-activated EC functions (proliferation, survival, migration, and tube formation). The alpha(9) and alpha(7) nAChRs have opposing effects on nicotine-induced cell proliferation and survival. Our studies reveal a critical role for the alpha(7) nAChR in mediating the effects of nicotine on the endothelium. Other subunits play a modulatory role. These findings may have therapeutic implications for diseases characterized by pathological angiogenesis.
View details for DOI 10.1002/jcb.22270
View details for Web of Science ID 000270438000012
View details for PubMedID 19623583
Naive CD4 T Cell Proliferation Is Controlled by Mammalian Target of Rapainaycin Regulation of GRAIL Expression
JOURNAL OF IMMUNOLOGY
2009; 182 (10): 5919-5928
In this study, we demonstrate that the E3 ubiquitin ligase gene related to anergy in lymphocytes (GRAIL) is expressed in quiescent naive mouse and human CD4 T cells and has a functional role in inhibiting naive T cell proliferation. Following TCR engagement, CD28 costimulation results in the expression of IL-2 whose signaling through its receptor activates the Akt-mammalian target of rapamycin (mTOR) pathway. Activation of mTOR allows selective mRNA translation, including the epistatic regulator of GRAIL, Otubain-1 (Otub1), whose expression results in the degradation of GRAIL and allows T cell proliferation. The activation of mTOR appears to be the critical component of IL-2R signaling regulating GRAIL expression. CTLA4-Ig treatment blocks CD28 costimulation and resultant IL-2 expression, whereas rapamycin and anti-IL-2 treatment block mTOR activation downstream of IL-2R signaling. Thus, all three of these biotherapeutics inhibit mTOR-dependent translation of mRNA transcripts, resulting in blockade of Otub1 expression, maintenance of GRAIL, and inhibition of CD4 T cell proliferation. These observations provide a mechanistic pathway sequentially linking CD28 costimulation, IL-2R signaling, and mTOR activation as important requirements for naive CD4 T cell proliferation through the regulation of Otub1 and GRAIL expression. Our findings also extend the role of GRAIL beyond anergy induction and maintenance, suggesting that endogenous GRAIL regulates general cell cycle and proliferation of primary naive CD4 T cells.
View details for DOI 10.4049/jimmunol.0803986
View details for Web of Science ID 000265899800008
View details for PubMedID 19414743
Type I interferon receptor controls B-cell expression of nucleic acid-sensing Toll-like receptors and autoantibody production in a murine model of lupus
ARTHRITIS RESEARCH & THERAPY
2009; 11 (4)
Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by the production of high-titer IgG autoantibodies directed against nuclear autoantigens. Type I interferon (IFN-I) has been shown to play a pathogenic role in this disease. In the current study, we characterized the role of the IFNAR2 chain of the type I IFN (IFN-I) receptor in the targeting of nucleic acid-associated autoantigens and in B-cell expression of the nucleic acid-sensing Toll-like receptors (TLRs), TLR7 and TLR9, in the pristane model of lupus.Wild-type (WT) and IFNAR2-/- mice were treated with pristane and monitored for proteinuria on a monthly basis. Autoantibody production was determined by autoantigen microarrays and confirmed using enzyme-linked immunosorbent assay (ELISA) and immunoprecipitation. Serum immunoglobulin isotype levels, as well as B-cell cytokine production in vitro, were quantified by ELISA. B-cell proliferation was measured by thymidine incorporation assay.Autoantigen microarray profiling revealed that pristane-treated IFNAR2-/- mice lacked autoantibodies directed against components of the RNA-associated autoantigen complexes Smith antigen/ribonucleoprotein (Sm/RNP) and ribosomal phosphoprotein P0 (RiboP). The level of IgG anti-single-stranded DNA and anti-histone autoantibodies in pristane-treated IFNAR2-/- mice was decreased compared to pristane-treated WT mice. TLR7 expression and activation by a TLR7 agonist were dramatically reduced in B cells from IFNAR2-/- mice. IFNAR2-/- B cells failed to upregulate TLR7 as well as TLR9 expression in response to IFN-I, and effector responses to TLR7 and TLR9 agonists were significantly decreased as compared to B cells from WT mice following treatment with IFN-alpha.Our studies provide a critical link between the IFN-I pathway and the regulation of TLR-specific B-cell responses in a murine model of SLE.
View details for DOI 10.1186/ar2771
View details for Web of Science ID 000270936400027
View details for PubMedID 19624844
- Regulation of GRAIL Expression by mTOR Controls Naive CD4 T Cell Proliferation ACADEMIC PRESS INC ELSEVIER SCIENCE. 2009: S35-S36
Protein microarrays with carbon nanotubes as multicolor Raman labels
2008; 26 (11): 1285-1292
The current sensitivity of standard fluorescence-based protein detection limits the use of protein arrays in research and clinical diagnosis. Here, we use functionalized, macromolecular single-walled carbon nanotubes (SWNTs) as multicolor Raman labels for highly sensitive, multiplexed protein detection in an arrayed format. Unlike fluorescence methods, Raman detection benefits from the sharp scattering peaks of SWNTs with minimal background interference, affording a high signal-to-noise ratio needed for ultra-sensitive detection. When combined with surface-enhanced Raman scattering substrates, the strong Raman intensity of SWNT tags affords protein detection sensitivity in sandwich assays down to 1 fM--a three-order-of-magnitude improvement over most reports of fluorescence-based detection. We use SWNT Raman tags to detect human autoantibodies against proteinase 3, a biomarker for the autoimmune disease Wegener's granulomatosis, diluted up to 10(7)-fold in 1% human serum. SWNT Raman tags are not subject to photobleaching or quenching. By conjugating different antibodies to pure (12)C and (13)C SWNT isotopes, we demonstrate multiplexed two-color SWNT Raman-based protein detection.
View details for DOI 10.1038/nbt.1501
View details for Web of Science ID 000260832200024
View details for PubMedID 18953353
HIT: a versatile proteomics platform for multianalyte phenotyping of cytokines, intracellular proteins and surface molecules
2008; 14 (11): 1284-1289
We have developed a multianalyte fluid-phase protein array technology termed high-throughput immunophenotyping using transcription (HIT). This method employs a panel of monoclonal antibodies, each tagged with a unique oligonucleotide sequence that serves as a molecular bar code. After staining a sample, T7 polymerase amplifies the tags, which are then hybridized to a DNA microarray for indirect measurement of each analyte. Although there are many potential applications for this technology, here we report its suitability for profiling cytokines, intracellular molecules and cell surface markers. Using HIT, we profiled 90 surface markers on human naive T helper cells activated in vitro. The markers identified in this screen are consistent with previously described activation markers and were validated by flow cytometry. Additionally, a HIT screen of surface markers expressed on T helper cells activated in the presence of transforming growth factor-beta identified downregulation of CD26 in these cells. HIT arrays are an ideal platform for rapidly identifying markers for further characterization and therapeutic intervention.
View details for DOI 10.1038/nm.1755
View details for Web of Science ID 000260751200049
View details for PubMedID 18849997
Expression-based Pathway Signature Analysis (EPSA): Mining publicly available microarray data for insight into human disease
BMC MEDICAL GENOMICS
Publicly available data repositories facilitate the sharing of an ever-increasing amount of microarray data. However, these datasets remain highly underutilized. Reutilizing the data could offer insights into questions and diseases entirely distinct from those considered in the original experimental design.We first analyzed microarray datasets derived from known perturbations of specific pathways using the samr package in R to identify specific patterns of change in gene expression. We refer to these pattern of gene expression alteration as a "pathway signatures." We then used Spearman's rank correlation coefficient, a non-parametric measure of correlation, to determine similarities between pathway signatures and disease profiles, and permutation analysis to evaluate false discovery rate. This enabled detection of statistically significant similarity between these pathway signatures and corresponding changes observed in human disease. Finally, we evaluated pathway activation, as indicated by correlation with the pathway signature, as a risk factor for poor prognosis using multiple unrelated, publicly available datasets.We have developed a novel method, Expression-based Pathway Signature Analysis (EPSA). We demonstrate that ESPA is a rigorous computational approach for statistically evaluating the degree of similarity between highly disparate sources of microarray expression data. We also show how EPSA can be used in a number of cases to stratify patients with differential disease prognosis. EPSA can be applied to many different types of datasets in spite of different platforms, different experimental designs, and different species. Applying this method can yield new insights into human disease progression.EPSA enables the use of publicly available data for an entirely new, translational purpose to enable the identification of potential pathways of dysregulation in human disease, as well as potential leads for therapeutic molecular targets.
View details for DOI 10.1186/1755-8794-1-51
View details for Web of Science ID 000272706500001
View details for PubMedID 18937865
Modulation of Peripheral B Cell Tolerance by CD72 in a Murine Model
ARTHRITIS AND RHEUMATISM
2008; 58 (10): 3192-3204
B cells play a dominant role in the pathogenesis of several autoimmune diseases, including systemic lupus erythematosus. It is not well understood how B cell signaling contributes to autoantibody production. The goal of this study was to elucidate the role of CD72 in modulating B cell receptor (BCR)-mediated tolerogenic signaling and peripheral B cell tolerance.A mouse model utilizing hen egg lysozyme (HEL) "anergic" B cells was studied. CD72-deficient mice carrying the BCR-specific IgHEL and/or soluble HEL (sHEL) transgenes were generated by breeding IgHEL-transgenic MD4 mice and/or sHEL-transgenic ML5 mice with congenic, CD72-deficient C57BL/6J mice. Normal and anergic B cells were isolated for analyses of B cell signaling. Aged wild-type and CD72-deficient mice were also examined for autoimmune phenomena.In the absence of CD72, anergic B cells inappropriately proliferated and survived in response to stimulation with self antigen. Biochemical analyses indicated that in anergic B cells, CD72 dominantly down-regulated BCR signaling to limit the antigen-induced elevation in [Ca2+]i and the activation of NFATc1, NF-kappaB, MAPK, and Akt. Mechanistically, CD72 was associated with, and regulated, the molecular adaptor Cbl-b in anergic B cells, suggesting that Cbl-b may play a role in mediating the negative effects of CD72 on BCR signaling. Moreover, in aged CD72-deficient mice, spontaneous production of antinuclear and anti-double-stranded DNA autoantibodies and features of lupus-like autoimmune disease were observed.CD72 is required to maintain B cell anergy and functions as a regulator of peripheral B cell tolerance. Thus, altered CD72 expression may play a role during the development of systemic lupus erythematosus.
View details for DOI 10.1002/art.23812
View details for Web of Science ID 000260024400029
View details for PubMedID 18821699
Evaluation of microarray surfaces and arraying parameters for autoantibody profiling
2008; 8 (17): 3443-3449
Autoantigen microarrays are being used increasingly to study autoimmunity. Significant variation has been observed when comparing microarray surfaces, printing methods, and probing conditions. In the present study, 24 surfaces and several arraying parameters were analyzed using >500 feature autoantigen microarrays printed with quill pins. A small subset of slides, including FAST, PATH, and SuperEpoxy2, performed well while maintaining the sensitivity and specificity of autoantigen microarrays previously demonstrated by our laboratory. By optimizing the major variables in our autoantigen microarray platform, subtle differences in serum samples can be identified that will shed light on disease pathogenesis.
View details for DOI 10.1002/pmic.200800146
View details for Web of Science ID 000259172400004
View details for PubMedID 18752214
Longitudinal comparison of B lymphocyte function from pristane-treated wild-type and IFNAR2-/- mice
WILEY-BLACKWELL. 2008: S316-S317
View details for Web of Science ID 000259244200422
Failure of oral atorvastatin to modulate a murine model of systemic lupus erythematosus
ARTHRITIS AND RHEUMATISM
2008; 58 (7): 2098-2104
Inhibitors of the 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase enzyme (statins) are cholesterol-lowering drugs that have shown promise as therapeutic agents in various animal models of autoimmune disease. The results of initial clinical trials with statins in multiple sclerosis and rheumatoid arthritis have also been encouraging. In this study, we attempted to treat a widely studied murine model of spontaneous systemic lupus erythematosus (SLE) with atorvastatin.(NZB x NZW)F1 (NZB/NZW) mice received daily oral doses of atorvastatin for 20 weeks. The mice were monitored weekly for survival and proteinuria. Anti-double-stranded DNA (anti-dsDNA) antibody levels in sera were determined by enzyme-linked immunosorbent assay (ELISA). T lymphocyte cytokine production in vitro, as well as cytokine levels in vivo, were measured by ELISA. T cell proliferation was assessed by thymidine incorporation assay. Serum cholesterol levels were determined using a standard fluorometric assay. Kidney tissue was harvested and evaluated for pathologic changes.In NZB/NZW mice, oral atorvastatin had significant effects on T cell proliferation and cytokine production in vitro. Atorvastatin also induced significant increases in serum levels of interleukin-4. However, atorvastatin treatment in NZB/NZW mice had no significant impact on proteinuria, survival, serum anti-dsDNA antibody and cholesterol levels, or extent of renal disease.Monotherapy with oral atorvastatin has no protective effects in a murine model of spontaneous SLE. The efficacy of atorvastatin in human SLE remains to be determined.
View details for DOI 10.1002/art.23605
View details for Web of Science ID 000257469800024
View details for PubMedID 18576356
Cytokines secreted in response to toll-like receptor ligand stimulation modulate differentiation of human Th17 cells
ARTHRITIS AND RHEUMATISM
2008; 58 (6): 1619-1629
Th17 cells (interleukin-17 [IL-17]-secreting T helper cells) have been implicated in the pathogenesis of rheumatoid arthritis and other autoimmune diseases, but the soluble factors that influence human Th17 differentiation have yet to be fully elucidated. This study was undertaken to investigate the hypothesis that the cytokines secreted by human peripheral blood mononuclear cells (PBMCs) in response to a subset of Toll-like receptor (TLR) ligands would influence Th17 polarization.Supernatants from human PBMCs treated with a panel of TLR agonists were tested for their ability to induce de novo IL-17 production in naive T helper cells. Multiplex cytokine analysis was used to identify candidate cytokines for subsequent blocking and sufficiency experiments.Conditioned media from PBMCs stimulated with TLR-4 or TLR-8/7 agonists, but not from those stimulated with TLR-2/1, -3, or -9 agonists, evoked robust secretion of IL-17 by T helper cells, independent of coculture with antigen-presenting cells. Multiplex analysis of 22 cytokines and chemokines identified a 6-factor cytokine signature that significantly correlated with IL-17-inducing activity. T cell activation in the presence of recombinant IL-1beta, IL-6, and IL-23 reconstituted robust IL-17 production, and this was enhanced by transforming growth factor beta (TGFbeta). IL-6 suppressed the expression of forkhead box P3 and reversed TGFbeta-mediated inhibition of T cell proliferation, but did not trigger IL-17 secretion. IL-17 production was completely abrogated by anti-IL-1 or IL-1 receptor antagonist and partially inhibited by anti-IL-6, anti-IL-2, or exogenous retinoic acid, but not by anti-tumor necrosis factor alpha. IL-1beta and IL-6 independently induced IL-21 secretion, but the presence of IL-21 alone was not sufficient for IL-17 production.These results indicate that ligation of a subset of TLRs generates proinflammatory cytokines that combine to potentiate human Th17 differentiation.
View details for DOI 10.1002/art.23497
View details for Web of Science ID 000256724900008
View details for PubMedID 18512782
- Protein microarrays address the elephant in the room CLINICAL CHEMISTRY 2008; 54 (6): 937-939
Peptide-coated nanotube-based biosensor for the detection of disease-specific autoantibodies in human serum
BIOSENSORS & BIOELECTRONICS
2008; 23 (10): 1413-1421
We demonstrate a label-free peptide-coated carbon nanotube-based immunosensor for the direct assay of human serum. A rheumatoid arthritis (RA)-specific (cyclic citrulline-containing) peptide, was immobilized to functionalized single-walled carbon nanotubes deposited on a quartz crystal microbalance (QCM) sensing crystal. Serum from RA patients was used to probe these nanotube-based sensors, and antibody binding was detected by QCM sensing. Specific antibody binding was also determined by comparing the assay of two serum control groups (normal and diseased sera), and the native unmodified peptide. The sensitivity of the nanotube-based sensor (detection in the femtomol range) was higher than that of the established ELISA and recently described microarray assay systems, detecting 34.4 and 37.5% more RA patients with anti-citrullinated peptide antibodies than those found by ELISA and microarray, respectively. There was also an 18.4 and 19.6% greater chance of a negative test being a true indicator of a person not having RA than by either ELISA or microarray, respectively. The performance of our label-free biosensor enables its application in the direct assay of sera in research and diagnostics.
View details for DOI 10.1016/j.bios.2007.11.022
View details for Web of Science ID 000255793200001
View details for PubMedID 18222083
Phase 2 trial of a DNA vaccine encoding myelin basic protein for multiple sclerosis
ANNALS OF NEUROLOGY
2008; 63 (5): 611-620
To evaluate the efficacy and safety of BHT-3009 in relapsing-remitting multiple sclerosis (MS) and to confirm that BHT-3009 causes immune tolerance.BHT-3009 is a tolerizing DNA vaccine for MS, encoding full-length human myelin basic protein. Relapsing-remitting MS patients were randomized 1:1:1 into three groups: placebo, 0.5 mg BHT-3009, or 1.5 mg BHT-3009, given intramuscularly at weeks 0, 2, 4, and every 4 weeks thereafter until week 44. The primary end point was the 4-week rate of occurrence of new gadolinium-enhancing lesions on brain magnetic resonance images from weeks 28 to 48. Protein microarrays were used to measure levels of anti-myelin autoantibodies.Compared with placebo, in the 267 patient analysis population the median 4-week rate of new enhancing lesions during weeks 28 to 48 was 50% lower with 0.5 mg BHT-3009 (p = 0.07) and during weeks 8 to 48 was 61% lower with 0.5 mg BHT-3009 (p = 0.05). The mean volume of enhancing lesions at week 48 was 51% lower on 0.5 mg BHT-3009 compared with placebo (p = 0.02). No significant improvement in magnetic resonance imaging lesion parameters was observed with 1.5 mg BHT-3009. Dramatic reductions in 23 myelin-specific autoantibodies in the 0.5 mg BHT-3009 arm were observed, but not with placebo or 1.5 mg BHT-3009.In relapsing-remitting MS patients, treatment with the lower dose (0.5 mg) of BHT-3009 for 44 weeks nearly attained the primary end point for reduction of the rate of new enhancing magnetic resonance imaging lesions (p = 0.07) and achieved several secondary end points including a reduction of the rate of enhancing magnetic resonance imaging lesions from weeks 8 to 48 (p = 0.05). Immunological data in a preselected subgroup of patients also indicated that treatment with 0.5 mg induced antigen-specific immune tolerance. The greater dose was ineffective.
View details for DOI 10.1002/ana.21370
View details for Web of Science ID 000255960600010
View details for PubMedID 18481290
IRF9 and STAT1 are required for IgG autoantibody production and B cell expression of TLR7 in mice
JOURNAL OF CLINICAL INVESTIGATION
2008; 118 (4): 1417-1426
A hallmark of SLE is the production of high-titer, high-affinity, isotype-switched IgG autoantibodies directed against nucleic acid-associated antigens. Several studies have established a role for both type I IFN (IFN-I) and the activation of TLRs by nucleic acid-associated autoantigens in the pathogenesis of this disease. Here, we demonstrate that 2 IFN-I signaling molecules, IFN regulatory factor 9 (IRF9) and STAT1, were required for the production of IgG autoantibodies in the pristane-induced mouse model of SLE. In addition, levels of IgM autoantibodies were increased in pristane-treated Irf9 -/- mice, suggesting that IRF9 plays a role in isotype switching in response to self antigens. Upregulation of TLR7 by IFN-alpha was greatly reduced in Irf9 -/- and Stat1 -/- B cells. Irf9 -/- B cells were incapable of being activated through TLR7, and Stat1 -/- B cells were impaired in activation through both TLR7 and TLR9. These data may reveal a novel role for IFN-I signaling molecules in both TLR-specific B cell responses and production of IgG autoantibodies directed against nucleic acid-associated autoantigens. Our results suggest that IFN-I is upstream of TLR signaling in the activation of autoreactive B cells in SLE.
View details for DOI 10.1172/JCI30065
View details for Web of Science ID 000254588600035
View details for PubMedID 18340381
RAG2 PHD finger couples histone H3 lysine 4 trimethylation with V(D)J recombination
2007; 450 (7172): 1106-U18
Nuclear processes such as transcription, DNA replication and recombination are dynamically regulated by chromatin structure. Eukaryotic transcription is known to be regulated by chromatin-associated proteins containing conserved protein domains that specifically recognize distinct covalent post-translational modifications on histones. However, it has been unclear whether similar mechanisms are involved in mammalian DNA recombination. Here we show that RAG2--an essential component of the RAG1/2 V(D)J recombinase, which mediates antigen-receptor gene assembly--contains a plant homeodomain (PHD) finger that specifically recognizes histone H3 trimethylated at lysine 4 (H3K4me3). The high-resolution crystal structure of the mouse RAG2 PHD finger bound to H3K4me3 reveals the molecular basis of H3K4me3-recognition by RAG2. Mutations that abrogate RAG2's recognition of H3K4me3 severely impair V(D)J recombination in vivo. Reducing the level of H3K4me3 similarly leads to a decrease in V(D)J recombination in vivo. Notably, a conserved tryptophan residue (W453) that constitutes a key structural component of the K4me3-binding surface and is essential for RAG2's recognition of H3K4me3 is mutated in patients with immunodeficiency syndromes. Together, our results identify a new function for histone methylation in mammalian DNA recombination. Furthermore, our results provide the first evidence indicating that disrupting the read-out of histone modifications can cause an inherited human disease.
View details for DOI 10.1038/nature06431
View details for Web of Science ID 000251579900092
View details for PubMedID 18033247
Free-solution oligonucleotide separation in nanoscale channels
2007; 79 (21): 8316-8322
In this paper, we report an experimental study of electrokinetic transport and separation of double-stranded deoxyribonucleic acid (dsDNA) oligonucleotides in custom-fabricated fused-silica nanochannels filled with a gel-free sodium borate aqueous buffer. Mixtures of fluorescently labeled dsDNA molecules in the range of 10-100 base pair (bp), fluorescein, and fluorescein-12-UTP (UTP) were separated in less than 120 s in channels of depth ranging from 40 to 1560 nm. We varied the channel depth and background buffer concentration to achieve a 0.006-0.2 range of Debye length-to-channel-half-depth ratio (lambdaD/h), and a 0.004-1.7 range of the ratio of length of dsDNA molecule to channel half-depth (l/h). We find observed oligonucleotide migration times depend on both l/h and lambdaD/h. Electrophoretic mobility estimates agree well with published (micrometer-scale channel) values for background electrolyte (BGE) concentrations greater than approximately 10 mM. At BGE concentrations of 1 and 5 mM, mobility estimates in our nanochannels are higher than published values. Of the cases studied, the highest separation sensitivities were achieved in 100 nm channels with 1-10 mM ion density buffers. Potential applications of this technology include rapid small-scale sequencing and other fluorescence-based oligonucleotide separation and detection assays.
View details for DOI 10.1021/ac0710580
View details for Web of Science ID 000250584800053
View details for PubMedID 17883279
Induction of antigen-specific tolerance in multiple sclerosis after immunization with DNA encoding myelin basic protein in a randomized, placebo-controlled phase 1/2 trial
ARCHIVES OF NEUROLOGY
2007; 64 (10): 1407-1415
To assess safety and immune modulation by BHT-3009, a tolerizing DNA vaccine encoding full-length human myelin basic protein, in patients with multiple sclerosis (MS).The study was a randomized, double-blind, placebo-controlled trial. Subjects receiving placebo were crossed over into an active arm after treatment unblinding.The trial was conducted at 4 academic institutions within North America. Patients Thirty patients with relapsing-remitting or secondary progressive MS who were not taking any other disease-modifying drugs were enrolled in the trial. Further, the patients were required to have either 1 to 5 gadolinium-enhancing lesions on screening brain magnetic resonance imaging (MRI), a relapse in the previous 2 years, or disease worsening in the previous 2 years.BHT-3009 was administered as intramuscular injections at weeks 1, 3, 5, and 9 after randomization into the trial, with or without 80 mg of daily oral atorvastatin calcium in combination. Three dose levels of BHT-3009 were tested (0.5 mg, 1.5 mg, and 3 mg).The primary outcome measures were safety and tolerability of BHT-3009. Secondary outcome measures included the number and volume of gadolinium-enhanced lesions on MRI, relapses, and analysis of antigen-specific immune responses.BHT-3009 was safe and well tolerated, provided favorable trends on brain MRI, and produced beneficial antigen-specific immune changes. These immune changes consisted of a marked decrease in proliferation of interferon-gamma-producing, myelin-reactive CD4+ T cells from peripheral blood and a reduction in titers of myelin-specific autoantibodies from cerebral spinal fluid as assessed by protein microarrays. We did not observe a substantial benefit of the atorvastatin combination compared with BHT-3009 alone.In patients with MS, BHT-3009 is safe and induces antigen-specific immune tolerance with concordant reduction of inflammatory lesions on brain MRI.
View details for Web of Science ID 000249998400004
View details for PubMedID 17698695
Notch signals positively regulate activity of the mTOR pathway in T-cell acute lymphoblastic leukemia
2007; 110 (1): 278-286
Constitutive Notch activation is required for the proliferation of a subgroup of T-cell acute lymphoblastic leukemia (T-ALL). Downstream pathways that transmit pro-oncogenic signals are not well characterized. To identify these pathways, protein microarrays were used to profile the phosphorylation state of 108 epitopes on 82 distinct signaling proteins in a panel of 13 T-cell leukemia cell lines treated with a gamma-secretase inhibitor (GSI) to inhibit Notch signals. The microarray screen detected GSI-induced hypophosphorylation of multiple signaling proteins in the mTOR pathway. This effect was rescued by expression of the intracellular domain of Notch and mimicked by dominant negative MAML1, confirming Notch specificity. Withdrawal of Notch signals prevented stimulation of the mTOR pathway by mitogenic factors. These findings collectively suggest that the mTOR pathway is positively regulated by Notch in T-ALL cells. The effect of GSI on the mTOR pathway was independent of changes in phosphatidylinositol-3 kinase and Akt activity, but was rescued by expression of c-Myc, a direct transcriptional target of Notch, implicating c-Myc as an intermediary between Notch and mTOR. T-ALL cell growth was suppressed in a highly synergistic manner by simultaneous treatment with the mTOR inhibitor rapamycin and GSI, which represents a rational drug combination for treating this aggressive human malignancy.
View details for DOI 10.1182/blood-2006-08-039883
View details for Web of Science ID 000247611000041
View details for PubMedID 17363738
Technology Insight: can autoantibody profiling improve clinical practice?
NATURE CLINICAL PRACTICE RHEUMATOLOGY
2007; 3 (2): 96-103
A hallmark of autoimmune diseases is the production of high titers of highly specific autoantibodies, which are routinely measured to guide clinical decision-making. Multiplex antigen microarrays are powerful tools that can provide profiles of the autoantibodies found in blood and other biological fluids. This high-throughput technology allows for rapid identification of antibody and antigen biomarker sets, which is sorely needed in the clinic to improve diagnosis, predictions of prognosis, and selection of targeted therapies. In this article we will describe the antigen microarray technologies that are currently available, and those that are in development. We highlight recent applications for antibody profiling, as well as the challenges that need to be faced before such technologies enter the clinic.
View details for DOI 10.1038/ncprheum0404
View details for Web of Science ID 000243966500010
View details for PubMedID 17299447
Proteome-wide analysis in Saccharomyces cerevisiae identifies several PHD fingers as novel direct and selective binding modules of histone H3 methylated at either lysine 4 or lysine 36
JOURNAL OF BIOLOGICAL CHEMISTRY
2007; 282 (4): 2450-2455
The PHD finger motif is a signature chromatin-associated motif that is found throughout eukaryotic proteomes. Here we have determined the histone methyl-lysine binding activity of the PHD fingers present within the Saccharomyces cerevisiae proteome. We provide evidence on the genomic scale that PHD fingers constitute a general class of effector modules for histone H3 trimethylated at lysine 4 (H3K4me3) and histone H3 trimethylated at lysine 36 (H3K36me3). Structural modeling of PHD fingers demonstrates a conserved mechanism for recognizing the trimethyl moiety and provides insight into the molecular basis of affinity for the different methyl-histone ligands. Together, our study suggests that a common function for PHD fingers is to transduce methyl-lysine events and sheds light on how a single histone modification can be linked to multiple biological outcomes.
View details for DOI 10.1074/jbc.C600286200
View details for Web of Science ID 000243593200036
View details for PubMedID 17142463
Role of MHC-linked genes in autoantigen selection and renal disease in a murine model of systemic lupus erythematosus
JOURNAL OF IMMUNOLOGY
2006; 177 (10): 7423-7434
We previously described a renal protective effect of factor B deficiency in MRL/lpr mice. Factor B is in the MHC cluster; thus, the deficient mice were H2b, the haplotype on which the knockout was derived, whereas the wild-type littermates were H2k, the H2 of MRL/lpr mice. To determine which protective effects were due to H2 vs factor B deficiency, we derived H2b congenic MRL/lpr mice from the 129/Sv (H2b) strain. Autoantibody profiling using autoantigen microarrays revealed that serum anti-Smith and anti-small nuclear ribonucleoprotein complex autoantibodies, while present in the majority of H2k/k MRL/lpr mice, were absent in the H2b/b MRL/lpr mice. Surprisingly, 70% of MRL/lpr H2b/b mice were found to be serum IgG3 deficient (with few to no IgG3-producing B cells). In addition, H2b/b IgG3-deficient MRL/lpr mice had significantly less proteinuria, decreased glomerular immune complex deposition, and absence of glomerular subepithelial deposits compared with MRL/lpr mice of any H2 type with detectable serum IgG3. Despite these differences, total histopathologic renal scores and survival were similar among the groups. These results indicate that genes encoded within or closely linked to the MHC region regulate autoantigen selection and isotype switching to IgG3 but have minimal effect on end-organ damage or survival in MRL/lpr mice.
View details for Web of Science ID 000242009700097
View details for PubMedID 17082662
Autoantigen arrays for multiplex analysis of antibody isotypes
2006; 6 (21): 5720-5724
We describe here a microarray-based method for multiplexed, antigen-specific assessment of immunoglobulin (Ig) subclasses. We used 1152-feature arrays composed of 140 antigens or antigen fragments to detect isotype-specific mAb, to quantitatively monitor changes in isotype mAb concentration, and to profile antigen-specific antibody isotype production in a murine model of autoimmunity. This platform can be easily adapted to a variety of applications, and has the potential to elucidate mechanisms that govern development and evolution of antibody responses in in vivo and in vitro systems.
View details for DOI 10.1002/pmic.200600345
View details for Web of Science ID 000242254600006
View details for PubMedID 17068762
Selective tyrosine kinase inhibition by imatinib mesylate for the treatment of autoimmune arthritis
JOURNAL OF CLINICAL INVESTIGATION
2006; 116 (10): 2633-2642
Tyrosine kinases play a central role in the activation of signal transduction pathways and cellular responses that mediate the pathogenesis of rheumatoid arthritis. Imatinib mesylate (imatinib) is a tyrosine kinase inhibitor developed to treat Bcr/Abl-expressing leukemias and subsequently found to treat c-Kit-expressing gastrointestinal stromal tumors. We demonstrate that imatinib potently prevents and treats murine collagen-induced arthritis (CIA). We further show that micromolar concentrations of imatinib abrogate multiple signal transduction pathways implicated in RA pathogenesis, including mast cell c-Kit signaling and TNF-alpha release, macrophage c-Fms activation and cytokine production, and fibroblast PDGFR signaling and proliferation. In our studies, imatinib attenuated PDGFR signaling in fibroblast-like synoviocytes (FLSs) and TNF-alpha production in synovial fluid mononuclear cells (SFMCs) derived from human RA patients. Imatinib-mediated inhibition of a spectrum of signal transduction pathways and the downstream pathogenic cellular responses may provide a powerful approach to treat RA and other inflammatory diseases.
View details for DOI 10.1172/JCI28546
View details for Web of Science ID 000240965700013
View details for PubMedID 16981009
A new two-color Fab labeling method for autoantigen protein microarrays
2006; 3 (9): 745-751
Antigen microarrays hold great promise for profiling the humoral immune response in the settings of autoimmunity, allergy and cancer. This approach involves immobilizing antigens on a slide surface and then exposing the array to biological fluids containing immunoglobulins. Although these arrays have proven extremely useful as research tools, they suffer from several sources of variability. To address these issues, we have developed a new two-color Fab labeling method that allows two samples to be applied simultaneously to the same array. This straightforward labeling approach improves reproducibility and reliably detects changes in autoantibody concentrations. Using this technique we profiled serum from a mouse model of systemic lupus erythematosus (SLE) and detected both expected and previously unrecognized reactivities. The improved labeling and detection method described here overcomes several problems that have hindered antigen microarrays and should facilitate translation to the clinical setting.
View details for DOI 10.1038/nmeth910
View details for Web of Science ID 000240290300020
View details for PubMedID 16929321
Single-cell analysis of siRNA-mediated gene silencing using multiparameter flow cytometry.
Cytometry. Part A : the journal of the International Society for Analytical Cytology
2006; 69 (2): 59-65
Use of synthetic short interfering RNAs (siRNAs) to study gene function has been limited by an inability to selectively analyze subsets of cells in complex populations, low and variable transfection efficiencies, and semiquantitative assays for measuring protein down-regulation. Intracellular flow cytometry can overcome these limitations by analyzing populations at the single-cell level in a high-throughput and quantitative fashion. Individual cells displaying a knockdown phenotype can be selectively interrogated for functional responses using multiparameter analysis.Lck-specific siRNA was delivered into Jurkat T cells or peripheral blood mononuclear cells (PBMCs) to suppress endogenous Lck expression. Transfected cells were fluorescently stained for intracellular Lck and analyzed using multiparameter flow cytometry. The Lck(lo) Jurkat subpopulation was selectively analyzed for CD69 up-regulation and phospho-states of signaling proteins following T-cell receptor (TCR) stimulation. Surface expression levels of CD4 and CD8 on transfected CD3+ gated PBMCs were correlated with intracellular Lck levels.A subpopulation of Jurkat cells with reduced levels of Lck was clearly resolved from cells with wildtype levels of Lck. Both CD69 up-regulation and ZAP70 phosphorylation were suppressed in Lck(lo) cells when compared with those in Lck(hi) cells upon TCR stimulation. Knockdown of intracellular Lck in primary T lymphocytes reduced surface expression of CD4 in a dose-dependent manner.Multiparameter flow cytometry is a powerful technique for the quantitative analysis of siRNA-mediated protein knockdown in complex hard-to-transfect cell populations.
View details for PubMedID 16419066
- Multiplexed protein array platformsfor analysis of autoimmune disease. Annu. Rev. Immunol. 2006; 24: 391-418
- Role of MHC- linked genes in autoantigen selection and renal disease in a murine model of SLE. Journal of Immunology 2006; in press
Detection of apoptosis-specific autoantibodies directed against granzyme B-induced cleavage fragments of the SS-B (La) autoantigen in sera from patients with primary Sjogren's syndrome
CLINICAL AND EXPERIMENTAL IMMUNOLOGY
2005; 142 (1): 148-154
The objective of this study was to detect autoantibodies against granzyme B cleavage products in sera from patients with primary Sjögren's syndrome (SS). Cell lysates derived from human salivary gland (HSG) cell lines were incubated with granzyme B. The susceptibility to the generation of cleavage fragments of SS autoantigens was assayed by immunoblotting using sera from 57 primary SS patients, 17 primary SS patients with malignant lymphoma (ML), 28 systemic lupus erythematosus (SLE) patients, and 20 healthy controls. A 27 kD protein was recognized by serum autoantibodies in 8 (14.0%) of 57 primary SS patients, 5 (29.4%) of 17 SS patients with ML, 2 (7.1%) of 28 SLE patients, but not in 20 normal subjects. This protein was recognized by anti-SSB (La) monoclonal antibodies. Granzyme B-treated recombinant La protein was also shown to migrate as a discrete 27 kD protein by SDS PAGE. Blocking studies demonstrated the existence of an apoptosis-specific B cell epitope present in sera from 2 of 8 primary SS patients and in 2 of 5 primary SS patients with ML which recognized the 27 kD protein. Granzyme B-induced La fragments are generated during cytotoxicity in vitro. This is the first report describing autoantibodies in sera from primary SS patients that specifically recognize fragments of the La protein that are produced by the granzyme B protease.
View details for DOI 10.1111/j.1365-2249.2005.02888.x
View details for Web of Science ID 000231824900020
View details for PubMedID 16178869
Sources of autoantigens in systemic lupus erythematosus
CURRENT OPINION IN RHEUMATOLOGY
2005; 17 (5): 513-517
A hallmark of systemic lupus erythematosus is the production of autoantibodies that recognize nuclear antigens. However, the underlying events and mechanisms that lead to the selection of these molecules for the autoimmune response remain poorly understood. In this review, we will examine some of the proposed explanations for sources of systemic lupus erythematosus-specific autoantigens. We will focus on events related to apoptosis, viral infection, cytokine production, innate immune system components, and alternative splicing of pre-mRNA transcripts.Definitive proof of a viral etiology for lupus remains elusive. However, recent observations have added to increasing evidence that viruses contribute to the bypass of tolerance in systemic lupus erythematosus. Also, events associated with apoptosis - most notably proteolytic autoantigen cleavage by caspases and granzyme B - have been implicated in the initiation of autoimmune responses for over a decade. Results obtained from animal models and human systems suggest complex functions for pro-apoptotic pathways in the regulation of immune responses. Inducible antigen expression and alternatively spliced transcripts may represent additional ways of generating autoantigenic material. Finally, toll-like receptor family members may play critical roles in the induction of antibody responses to nucleic acids in systemic lupus erythematosus.Several factors may contribute to the generation of systemic lupus erythematosus-specific autoantigens. Determining the underlying causes of autoantibody production may provide important insight into the etiology and pathogenesis of this disease.
View details for Web of Science ID 000231382800002
View details for PubMedID 16093826
Antigen microarray profiling of autoantibodies in rheumatoid arthritis
ARTHRITIS AND RHEUMATISM
2005; 52 (9): 2645-2655
Because rheumatoid arthritis (RA) is a heterogeneous autoimmune disease in terms of disease manifestations, clinical outcomes, and therapeutic responses, we developed and applied a novel antigen microarray technology to identify distinct serum antibody profiles in patients with RA.Synovial proteome microarrays, containing 225 peptides and proteins that represent candidate and control antigens, were developed. These arrays were used to profile autoantibodies in randomly selected sera from 2 different cohorts of patients: the Stanford Arthritis Center inception cohort, comprising 18 patients with established RA and 38 controls, and the Arthritis, Rheumatism, and Aging Medical Information System cohort, comprising 58 patients with a clinical diagnosis of RA of <6 months duration. Data were analyzed using the significance analysis of microarrays algorithm, the prediction analysis of microarrays algorithm, and Cluster software.Antigen microarrays demonstrated that autoreactive B cell responses targeting citrullinated epitopes were present in a subset of patients with early RA with features predictive of the development of severe RA. In contrast, autoimmune targeting of the native epitopes contained on synovial arrays, including several human cartilage gp39 peptides and type II collagen, were associated with features predictive of less severe RA.Proteomic analysis of autoantibody reactivities provides diagnostic information and allows stratification of patients with early RA into clinically relevant disease subsets.
View details for DOI 10.1002/art.21269
View details for Web of Science ID 000232115700009
View details for PubMedID 16142722
An array of possibilities for the study of autoimmunity
2005; 435 (7042): 605-611
Since the completion of the sequencing of the human genome, scientific focus has shifted from studying genes to analysing the much larger number of proteins encoded by them. Several proteins can be generated from a single gene depending on how the genetic information is read (transcribed) and how the resultant protein is modified following translation (post-translational modification). Genomic and proteomic technologies are already providing useful information about autoimmune disease, and they are likely to lead to important discoveries within the next decade.
View details for DOI 10.1038/nature03726
View details for Web of Science ID 000229476200038
View details for PubMedID 15931213
Granzyme B is dispensable for immunologic tolerance to self in a murine model of systemic lupus erythematosus
ARTHRITIS AND RHEUMATISM
2005; 52 (6): 1684-1693
Proteolytic autoantigen cleavage by the serine protease granzyme B has been implicated in the development of systemic autoimmune disease; however, there has been no conclusive demonstration of a pathogenic role for granzyme B in autoimmunity. In this study, we evaluated the role of granzyme B in a murine model of autoimmunity.To identify potential novel granzyme B substrates, complementary DNAs encoding nuclear factor 45 (NF45) and NF90 were used to generate (35)S-methionine-labeled proteins by coupled in vitro transcription/translation. Radiolabeled proteins were then incubated with purified recombinant granzyme B or caspases, and the cleavage products were analyzed by autoradiography. We also immunized granzyme B-deficient and granzyme B-intact mice with the mineral oil pristane. Production of autoantibodies directed against granzyme B substrates in response to pristane was evaluated by Western blotting, immunoprecipitation, and enzyme-linked immunosorbent assay.The double-stranded RNA-binding protein NF90 was identified as a novel substrate for caspases and granzyme B, both in vitro and in vivo. NF90 is uniquely cleaved by granzyme B in vitro; however, pristane immunization still induced anti-NF90 antibodies in granzyme B-deficient mice. Pristane-treated granzyme B-deficient mice also produced antibodies directed against the U1-70-kd antigen, a previously identified granzyme B substrate. Last, antibodies directed against U1-70 kd arose spontaneously in granzyme B-deficient mice.These results demonstrate that granzyme B is not required for the production of autoantibodies directed against antigens that are granzyme B substrates in vitro. The data also suggest a protective role for this proapoptotic protease in systemic autoimmunity.
View details for DOI 10.1002/art.21092
View details for Web of Science ID 000229721700012
View details for PubMedID 15934098
Protein arrays for studying blood cells and their secreted products
2005; 204: 264-282
Protein microarrays have been developed and partially validated for studying blood cells, which play a role in many human diseases. Arrays of capture antibodies are commercially available for analyzing cytokines and intracellular signaling proteins. Several academic laboratories have developed antigen microarrays for characterizing autoimmune and allergic diseases, with a goal toward using such arrays to profile antibodies found in blood or other biological fluids. Arrays composed of major histocompatibility complex tetramers have been constructed and validated for analysis of immune responses in mice, paving the way toward studying antigen-specific T-lymphocyte responses. Finally, reverse-phase protein lysate microarray technology, first developed for analyzing cancer cells from tissue sections, has now been demonstrated for studying living cells, including knockout cells, cells treated with drugs such as kinase inhibitors, and rare populations of lymphocytes such as regulatory T cells. The goal of this review is to focus on advances in and future uses of arrays of proteins that can be printed on glass microscope slides using traditional microarray robots that are commonly found at academic medical centers. Dissemination of protein array technology will occur in the next decade and will markedly change how immunology research, particularly in the fields of autoimmunity and inflammation, is conducted.
View details for Web of Science ID 000227672500019
View details for PubMedID 15790364
The challenge of analyzing the proteome in humans with autoimmune diseases
HUMAN IMMUNOLOGY: PATIENT-BASED RESEARCH
2005; 1062: 61-68
Analysis of blood samples from patients suffering from autoimmune diseases remains a mainstay in the clinic for initial diagnosis, prognostication, and clinical decision making. In particular, testing for the presence of serum autoantibodies has proved to be one of the most useful confirmatory assays for many different diseases. Recent genomic and transcript profiling studies have implicated certain cytokines, surface receptors, signaling pathways, and cell types in the pathogenesis of inflammatory diseases. The next obvious step is to delve into the much more complex level that follows the genome and transcriptome-the expressed proteome. This review focuses on several proteomics technologies being applied and/or developed by our laboratory for the study of autoimmunity, cancer, and cardiovascular disease, all of which are known to be associated with defects in immunity and inflammation. The findings of other participants in the recent Human Immunology Conference hosted by the Dana Foundation and the New York Academy of Sciences (May 17 & 18, 2005) are included. In particular, major pitfalls in the study of the human proteome are pointed out, and important areas for immediate investigation to move the field forward as rapidly as possible are proposed.
View details for DOI 10.1196/annals.1358.009
View details for Web of Science ID 000236473100007
View details for PubMedID 16461789
Suppression of autoimmunity via microbial mimics of altered peptide ligands
MOLECULAR MIMICRY: INFECTION-INDUCING AUTOIMMUNE DISEASE
2005; 296: 55-63
Molecular mimics of self-antigens can behave as altered peptide ligands and serve to ameliorate autoimmune disease. Analysis of experimental autoimmune encephalomyelitis with proteomic autoantibody microarrays reveals that there might exist a wide variety of microbes with features that mimic self-epitopes. Autoimmunity could therefore be modulated via microbial immunity, which may account for relapse and remission of ongoing disease.
View details for Web of Science ID 000235217000004
View details for PubMedID 16323420
- Granzyme B and natural killer (NK) death Modern Rheumatology 2005; 15: 315-322.
Protein microarrays for multiplex analysis of signal transduction pathways
2004; 10 (12): 1390-1396
We have developed a multiplexed reverse phase protein (RPP) microarray platform for simultaneous monitoring of site-specific phosphorylation of numerous signaling proteins using nanogram amounts of lysates derived from stimulated living cells. We first show the application of RPP microarrays to the study of signaling kinetics and pathway delineation in Jurkat T lymphocytes. RPP microarrays were used to profile the phosphorylation state of 62 signaling components in Jurkat T cells stimulated through their membrane CD3 and CD28 receptors, identifying a previously unrecognized link between CD3 crosslinking and dephosphorylation of Raf-1 at Ser259. Finally, the potential of this technology to analyze rare primary cell populations is shown in a study of differential STAT protein phosphorylation in interleukin (IL)-2-stimulated CD4(+)CD25(+) regulatory T cells. RPP microarrays, prepared using simple procedures and standard microarray equipment, represent a powerful new tool for the study of signal transduction in both health and disease.
View details for DOI 10.1038/nm1139
View details for Web of Science ID 000225500900035
View details for PubMedID 15558056
Murine CD4(+) CD25(+) regulatory T cells fail to undergo chromatin remodeling across the proximal promoter region of the IL-2 gene
JOURNAL OF IMMUNOLOGY
2004; 173 (8): 4994-5001
CD4+CD25+ regulatory T cells (Treg) acquire unique immunosuppressive properties while maintaining an anergy phenotype when activated in vitro under conditions that induce IL-2 production and proliferation in conventional CD4+ T cells. We investigated the mechanism underlying one component of this naturally anergic phenotype, the inability of the Treg cells to produce IL-2 following activation. Analysis of freshly isolated murine CD4+CD25+ Treg and conventional CD4+CD25- T cells following PMA/ionomycin stimulation demonstrated no differences in inducible AP-1 formation, an important transcriptional complex in regulating IL-2 gene expression. Although p38 MAPK and ERK1/2 protein kinases were phosphorylated with similar kinetics, we observed diminished activation of JNK in the CD4+CD25+ Treg cells. However, lentiviral-mediated reconstitution of the JNK pathway using a constitutively active construct did not overcome the block in IL-2 synthesis. Using a PCR-based chromatin accessibility assay we found that the minimal IL-2 promoter region of CD4+CD25+ Treg cells, unlike conventional CD4 T cells, did not undergo chromatin remodeling following stimulation, suggesting that the inability of CD4+CD25+ Treg cells to secrete IL-2 following activation is controlled at the chromatin level.
View details for Web of Science ID 000224392200028
View details for PubMedID 15470042
Interferon-alpha-inducible proteins are novel autoantigens in murine lupus
ARTHRITIS AND RHEUMATISM
2004; 50 (10): 3239-3249
To investigate the spectrum of B cell autoimmunity in the recently described anti-CD1-autoreactive T cell receptor (TCR)-transgenic murine lupus-like (CD1 lupus-like) model.Lethally irradiated BALB/c/nu/nu mice were injected intravenously with donor BALB/c bone marrow and spleen cells expressing TCRalpha and TCRbeta transgenes that recognize CD1d. Sera from adoptive host animals that developed lupus (i.e., CD1 lupus mice) were collected at serial time points and analyzed by Western blotting and immunoprecipitation, using protein extracts prepared from NIH3T3 mouse fibroblasts and EL-4 lymphocytes, respectively. Sera obtained from older animals in several models of spontaneous lupus (NZB/NZW, MRL++, and MRL/lpr mice), unmanipulated BALB/c/nu/nu mice, and normal BALB/c mice were used as controls.Analyses demonstrated that the prominent targets of autoantibodies in the CD1 lupus-like model are interferon-alpha (IFNalpha)-inducible antigens. Biochemical and serologic characterizations identified one antigen as belonging to the interferon-inducible 202 (Ifi202) subfamily of proteins within the Ifi200 family, and a second antigen as a member of the 70-kd heat-shock protein family. Autoantibodies directed against these antigens were rapidly produced at an early stage of disease. Anti-p50 autoantibodies were present in sera from 7 (78%) of 9 CD1 lupus mice that developed severe kidney disease.IFNalpha-inducible proteins represent a novel class of autoantigens in murine lupus, and the findings suggest additional roles for IFNalpha in this disease. Since Ifi202 autoantigens are encoded by the murine non-major histocompatibility complex lupus-susceptibility gene locus Ifi202, these data provide a link between recent advances in lupus genetics and the formation of autoantibodies.
View details for DOI 10.1002/art.20508
View details for Web of Science ID 000224508400023
View details for PubMedID 15476221
Characterization of novel antigens recognized by serum autoantibodies from anti-CD1 TCR-transgenic lupus mice
EUROPEAN JOURNAL OF IMMUNOLOGY
2004; 34 (6): 1654-1662
In this study, we further characterize the humoral autoimmune response in the recently described anti-CD1 autoreactive T cell receptor-transgenic mouse lupus model (CD1 lupus model). We discovered and characterized novel autoantigens, comprising a protein of 105 kDa (p105) and a novel RNA molecule of 140 base pairs (bp) that is likely associated with p105, and several additional factors with distinct biochemical properties. In the CD1 lupus model, lethally irradiated BALB/c/nu/nu mice were injected intravenously with sorted bone marrow cells and sorted splenic T cells from donor BALB/c mice expressing TCR alpha and beta transgenes that encode autoreactivity for CD1d. Adoptive hosts injected with the single-positive (CD4(+) and CD8(+)) subset of transgenic cells developed anti-double-stranded DNA antibodies and a lupus-like illness. Sera were analyzed by Western blotting and immunoprecipitation. Antigens were characterized by biochemical and serological methods. Serum autoantibodies from 5 of 12 (42%) CD1 lupus mice immunoprecipitated a 105-kDa protein, termed p105. p105 was associated with a small RNA of approximately 140 bp. Anti-p105 autoantibodies appeared early in the course of disease. Serological and biochemical characterization suggested that p105 was distinct from known lupus autoantigens of similar molecular masses, indicating that p105 represents a novel autoantigen in lupus.
View details for DOI 10.1002/eji.200324201
View details for Web of Science ID 000221993200017
View details for PubMedID 15162435
Proteolytic cleavage of the catalytic subunit of DNA-dependent protein kinase during poliovirus infection
JOURNAL OF VIROLOGY
2004; 78 (12): 6313-6321
DNA-dependent protein kinase (DNA-PK) is a serine/threonine kinase that has critical roles in DNA double-strand break repair, as well as B- and T-cell antigen receptor rearrangement. The DNA-PK enzyme consists of the Ku regulatory subunit and a 450-kDa catalytic subunit termed DNA-PK(CS). Both of these subunits are autoantigens associated with connective tissue diseases such as systemic lupus erythematosus (SLE) and scleroderma. In this report, we show that DNA-PK(CS) is cleaved during poliovirus infection of HeLa cells. Cleavage was visible as early as 1.5 h postinfection (hpi) and resulted in an approximately 40% reduction in the levels of native protein by 5.5 hpi. Consistent with this observation, the activity of the DNA-PK(CS) enzyme was also reduced during viral infection, as determined by immunoprecipitation kinase assays. Although it has previously been shown that DNA-PK(CS) is a substrate of caspase-3 in vitro, the protein was still cleaved during poliovirus infection of the caspase-3-deficient MCF-7 cell line. Cleavage was not prevented by infection in the presence of a soluble caspase inhibitor, suggesting that cleavage in vivo was independent of host caspase activation. DNA-PK(CS) is directly cleaved by a picornaviral 2A protease in vitro, producing a fragment similar in size to the cleavage product observed in vivo. Taken together, our results indicate that DNA-PK(CS) is cleaved by the 2A protease during poliovirus infection. Proteolytic cleavage of DNA-PK(CS) during poliovirus infection may contribute to inhibition of host immune responses. Furthermore, cleavage of autoantigens by viral proteases may target these proteins for the autoimmune response by generating novel, or "immunocryptic," protein fragments.
View details for DOI 10.1128/JVI.78.12.6313-6321.2004
View details for Web of Science ID 000221772000025
View details for PubMedID 15163725
High-throughput methods for measuring autoantibodies in systemic lupus erythematosus and other autoimmune diseases
2004; 37 (4): 269-272
Numerous groups have now validated high-throughput approaches to autoantibody profiling in a variety of systems. Recently, we have used autoantigen microarray technology to identify distinct autoantibody profiles in H-2 congenic MRL/lpr mice (Sekine et al., manuscript in preparation), and we are expanding this platform to study human and mouse models of IDDM and RA. We are also developing protein arrays for multiplex analysis of serum antibody isotypes. Multiplexed methods for autoantibody profiling will undoubtedly continue to uncover novel aspects of autoimmunity and B cell biology. It is now time to move these technologies beyond the proof-of-concept phase, and start addressing the next series of important questions. These include, but certainly are not limited to: identifying "autoantibody signatures" associated with disease state or outcome; profiling autoantibodies during the natural course of murine and human disease; and monitoring changes in autoantibody profiles of patients in response to therapeutic intervention. However, the next set of challenges is just right around the corner. As data and statistical analysis tools become more robust, it will be possible to generate and approach new hypotheses at an unprecedented pace.
View details for DOI 10.1080/08916930410001710686
View details for Web of Science ID 000223372000006
View details for PubMedID 15518040
- "hot technologies" for clinical immunology research CLINICAL IMMUNOLOGY 2004; 111 (2): 153-154
- Unlocking the "PAD" lock on rheumatoid arthritis ANNALS OF THE RHEUMATIC DISEASES 2004; 63 (4): 330-332
Antibodies in scleroderma: direct pathogenicity and phenotypic associations.
Current rheumatology reports
2004; 6 (2): 156-163
Scleroderma is an autoimmune disease involving endothelial cell damage and fibroblast overproduction of extracellular matrix. Several autoantibodies present in the sera of patients with scleroderma, including anti-endothelial cell, antifibroblast, anti-matrix metalloproteinase, and antifibrillin-1 antibodies, may directly contribute to disease pathogenesis. Scleroderma also is characterized by the presence of antinuclear and antinucleolar antibodies, which correlate with particular phenotypes. These include antitopoisomerase-I, anticentromere, antihistone, anti-polymyositis/scleroderma, anti-Th/To, anti-U3-small nucleolar ribonucleoprotein particle, anti-U1-small nuclear ribonucleoprotein particle, anti-RNA polymerase, and anti-B23 antibodies. Other antibodies classically associated with other autoimmune diseases, such as antiphospholipid, antineutrophil cytoplasmic, and antimitochondrial antibodies, also have been described in patients with scleroderma. This review will summarize the various autoantibodies associated with scleroderma, their putative pathogenic roles, and their phenotypic correlations.
View details for PubMedID 15016347
Multiplexed assays for identification of biomarkers and surrogate markers in systemic lupus erythematosus
2004; 13 (5): 304-311
Validated biomarkers and surrogate markers are badly needed for monitoring patients with systemic lupus erythematosus (SLE), both for routine clinical care and for clinical trials research. SLE is difficult to study in clinical trials, in part because the disease is so heterogeneous. Very few useful markers have been identified, and even those that historically have been thought to be valid have been recently questioned. This report will focus on the use of emerging multiplexed assay formats that enable analysis of hundreds or even thousands of analytes simultaneously. Their potential and pitfalls for monitoring patients with SLE, particularly those enrolled in clinical trials testing novel therapeutics, will be discussed.
View details for Web of Science ID 000222152800004
View details for PubMedID 15230283
Protein arrays for autoantibody profiling and fine-specificity mapping
2003; 3 (11): 2077-2084
Protein arrays provide a powerful approach to study autoimmune disease. Autoimmune responses activate B cells to produce autoantibodies that recognize self-molecules termed autoantigens, many of which are proteins or protein complexes. Protein arrays enable profiling of the specificity of autoantibody responses against panels of peptides and proteins representing known autoantigens as well as candidate autoantigens. In addition to identifying autoantigens and mapping immunodominant epitopes, proteomic analysis of autoantibody responses will further enable diagnosis, prognosis, and tailoring of antigen-specific tolerizing therapy.
View details for DOI 10.1002/pmic.200300583
View details for Web of Science ID 000186582500002
View details for PubMedID 14595805
Protein microarrays guide tolerizing DNA vaccine treatment of autoimmune encephalomyelitis
2003; 21 (9): 1033-1039
The diversity of autoimmune responses poses a formidable challenge to the development of antigen-specific tolerizing therapy. We developed 'myelin proteome' microarrays to profile the evolution of autoantibody responses in experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis (MS). Increased diversity of autoantibody responses in acute EAE predicted a more severe clinical course. Chronic EAE was associated with previously undescribed extensive intra- and intermolecular epitope spreading of autoreactive B-cell responses. Array analysis of autoantigens targeted in acute EAE was used to guide the choice of autoantigen cDNAs to be incorporated into expression plasmids so as to generate tolerizing vaccines. Tolerizing DNA vaccines encoding a greater number of array-determined myelin targets proved superior in treating established EAE and reduced epitope spreading of autoreactive B-cell responses. Proteomic monitoring of autoantibody responses provides a useful approach to monitor autoimmune disease and to develop and tailor disease- and patient-specific tolerizing DNA vaccines.
View details for DOI 10.1038/nbt859
View details for Web of Science ID 000185051000035
View details for PubMedID 12910246
Synovial proteome microarrays identify deiminated proteins as targets of the autoantibody response in Rheumatoid Arthritis
WILEY-BLACKWELL. 2003: S429-S429
View details for Web of Science ID 000185432801113
Autoantibodies in early arthritis: Advances in diagnosis and prognostication
CLINICAL AND EXPERIMENTAL RHEUMATOLOGY
2003; 21 (5): S59-S64
Several excellent reviews have recently been published on the significance of autoantibodies in rheumatoid arthritis (RA) (1-4). Here we: (i) review selected longitudinal studies examining the predictive utility of autoantibodies in early arthritis and early RA cohorts; (ii) assess the relevance of autoantibodies as an independent parameter for prediction and prognostication of RA; and (iii) describe the potential of multiplex autoantibody assays, including miniaturized, high-throughput microarray technology, to improve diagnosis and prognostication in recent-onset synovitis/early arthritis patients.
View details for Web of Science ID 000185970100011
View details for PubMedID 14969052
Noncovalent functionalization of carbon nanotubes for highly specific electronic biosensors
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2003; 100 (9): 4984-4989
Novel nanomaterials for bioassay applications represent a rapidly progressing field of nanotechnology and nanobiotechnology. Here, we present an exploration of single-walled carbon nanotubes as a platform for investigating surface-protein and protein-protein binding and developing highly specific electronic biomolecule detectors. Nonspecific binding on nanotubes, a phenomenon found with a wide range of proteins, is overcome by immobilization of polyethylene oxide chains. A general approach is then advanced to enable the selective recognition and binding of target proteins by conjugation of their specific receptors to polyethylene oxide-functionalized nanotubes. This scheme, combined with the sensitivity of nanotube electronic devices, enables highly specific electronic sensors for detecting clinically important biomolecules such as antibodies associated with human autoimmune diseases.
View details for DOI 10.1073/pnas.0837064100
View details for Web of Science ID 000182612600006
View details for PubMedID 12697899
On-chip coupling of isoelectric focusing and free solution electrophoresis for multidimensional separations
2003; 75 (5): 1180-1187
We have developed an acrylic microfluidic device that sequentially couples liquid-phase isoelectric focusing (IEF) and free solution capillary electrophoresis (CE). Rapid separation (<1 min) and preconcentration (73x) of species were achieved in the initial IEF dimension. Using full-field fluorescence imaging, we observed nondispersive mobilization velocities on the order of 20 microm/s during characterization of the IEF step. This transport behavior allowed controlled electrokinetic mobilization of focused sample bands to a channel junction, where voltage switching was used to repeatedly inject effluent from the IEF dimension into an ampholyte-based CE separation. This second dimension was capable of analyzing all fluid volumes of interest from the IEF dimension, as IEF was 'parked' during each CE analysis and refocused prior to additional CE analyses. Investigation of each dimension of the integrated system showed time-dependent species displacement and band-broadening behavior consistent with IEF and CE, respectively. The peak capacity of the 2D system was approximately 1300. A comprehensive 2D analysis of a fluid volume spanning 15% of the total IEF channel length was completed in less than 5 min.
View details for DOI 10.1021/ac026239a
View details for Web of Science ID 000181259300027
View details for PubMedID 12641239
- Interpreting interest in interferon-alpha ARTHRITIS RESEARCH & THERAPY 2003; 5 (5): 246-248
- Multiplex autoantibody profiling using 'synovial proteome' microarrays identifies citrulline-modified peptides as major targets of the autoimmune response in rheumatoid arthritis BIOMED CENTRAL LTD. 2003: S31-S31
Protein and peptide array analysis of autoimmune disease
Molecular cloning, sequencing of the human genome, and other major advances in biomedical research have contributed substantially to our understanding of autoimmune disease. Nevertheless, to date, such advances have failed to reveal the etiology of or yield curative therapies for autoimmune disease. New approaches are needed. Proteomics, the large-scale study of expression and function of proteins that compose our tissues and mediate disease, represents a powerful and promising strategy. We developed protein and peptide arrays to profile autoantibody responses in autoimmune disease. Protein and peptide array analysis of autoimmune samples is revealing human and pathogen proteins involved in initiation and perpetuation of autoimmunity. Proteomic determination of autoantibody profiles can be utilized for diagnosis, prognostication, and guiding tolerizing therapy for autoimmune disease.
View details for Web of Science ID 000180064700010
View details for PubMedID 12514932
Human autoimmune sera as molecular probes for the identification of an autoantigen kinase signaling pathway
JOURNAL OF EXPERIMENTAL MEDICINE
2002; 196 (9): 1213-1225
Using human autoimmune sera as molecular probes, we previously described the association of phosphorylated serine/arginine splicing factors (SR splicing factors) with the U1-small nuclear ribonucleoprotein (U1-snRNP) and U3-small nucleolar RNP (snoRNP) in apoptotic cells. SR proteins are highly conserved autoantigens whose activity is tightly regulated by reversible phosphorylation of serine residues by at least eight different SR protein kinase kinases (SRPKs), including SRPK1, SRPK2, and the scleroderma autoantigen topoisomerase I. In this report, we demonstrate that only one of the known SRPKs, SRPK1, is associated with the U1-snRNP autoantigen complex in healthy and apoptotic cells. SRPK1 is activated early during apoptosis, followed by caspase-mediated proteolytic inactivation at later time points. SRPKs are cleaved in vivo after multiple apoptotic stimuli, and cleavage can be inhibited by overexpression of bcl-2 and bcl-x(L), and by exposure to soluble peptide caspase inhibitors. Incubation of recombinant caspases with in vitro-translated SRPKs demonstrates that SRPK1 and SRPK2 are in vitro substrates for caspases-8 and -9, respectively. In contrast, topoisomerase I is cleaved by downstream caspases (-3 and -6). Since each of these SRPKs sits at a distinct checkpoint in the caspase cascade, SRPKs may serve an important role in signaling pathways governing apoptosis, alternative mRNA splicing, SR protein trafficking, RNA stability, and possibly the generation of autoantibodies directed against splicing factors.
View details for DOI 10.1084/jem.20021167
View details for Web of Science ID 000179151300009
View details for PubMedID 12417631
Millennium Award. Proteomics for the development of DNA tolerizing vaccines to treat autoimmune disease.
2002; 103 (1): 7-12
Autoimmune disease affects 3% of the world population, yet current therapies that globally suppress immune function are inadequate. Tremendous need exists for specific and curative therapies, and we describe a strategy for development of antigen-specific therapies that inactivate pathogenic lymphocytes causing tissue injury. Major barriers to development of antigen-specific therapies for T-cell-mediated autoimmune diseases, such as multiple sclerosis, rheumatoid arthritis, and autoimmune diabetes, include (i) lack of knowledge of the specificity of autoimmune responses, for which proteomic technologies represent powerful tools to identify the self-protein targets of the autoimmune response, and (ii) lack of methods to induce specific immune tolerance, for which DNA tolerizing vaccines represent a promising strategy. We termed our approach Reverse Genomics: use of the proteomics-determined specificity of the autoantibody response to develop and select DNA tolerizing vaccines. Studies performed using animal models for multiple sclerosis and autoimmune diabetes support our Reverse Genomics approach. Through integration of proteomics with specific tolerizing therapies, we are developing a comprehensive approach to treat human autoimmune disease.
View details for PubMedID 11987980
- Proteomics technologies for the study of autoimmune disease ARTHRITIS AND RHEUMATISM 2002; 46 (4): 885-893
Autoantigen microarrays for multiplex characterization of autoantibody responses
2002; 8 (3): 295-301
We constructed miniaturized autoantigen arrays to perform large-scale multiplex characterization of autoantibody responses directed against structurally diverse autoantigens, using submicroliter quantities of clinical samples. Autoantigen microarrays were produced by attaching hundreds of proteins, peptides and other biomolecules to the surface of derivatized glass slides using a robotic arrayer. Arrays were incubated with patient serum, and spectrally resolvable fluorescent labels were used to detect autoantibody binding to specific autoantigens on the array. We describe and characterize arrays containing the major autoantigens in eight distinct human autoimmune diseases, including systemic lupus erythematosus and rheumatoid arthritis. This represents the first report of application of such technology to multiple human disease sera, and will enable validated detection of antibodies recognizing autoantigens including proteins, peptides, enzyme complexes, ribonucleoprotein complexes, DNA and post-translationally modified antigens. Autoantigen microarrays represent a powerful tool to study the specificity and pathogenesis of autoantibody responses, and to identify and define relevant autoantigens in human autoimmune diseases.
View details for Web of Science ID 000174139500036
View details for PubMedID 11875502
21st European Workshop for Rheumatology Research, Vienna, Austria, 1-4 March 2001.
Major advances in technology now drive how we approach questions in immunology, particularly in analyzing complex data sets commonly encountered in genomics and proteomics studies. Active areas of investigation include development of novel technologies, identification of elusive target antigens for RA and other diseases, dissection of signaling pathways connecting the lymphocyte cell surface with the nucleus, and exploration of new avenues for therapeutic interventions. The European Workshop for Rheumatology Research (EWRR) is a forum for many European and non-European scientists to present research findings of high quality. Arthritis researchers from around the globe should be strongly encouraged to attend future meetings, the next of which is the 22nd EWRR meeting in Leiden, the Netherlands, in 2002.
View details for PubMedID 11438041
Life and death decisions: regulation of apoptosis by proteolysis of signaling molecules
CELL DEATH AND DIFFERENTIATION
2000; 7 (7): 589-602
Caspases are the major executioners of cell death, serving as molecular guillotines to behead many proteins required for maintenance of cellular homeostasis. Identification of caspase substrates has taken on increasing importance as we attempt to better understand the molecular mechanisms involved in regulating the struggle between life and death. Many caspase substrates have been described and include RNA binding proteins such as La and U1-70 kD, structural proteins such as keratin and nuclear lamins, and transcription factors or their regulatory proteins that include IkappaB, SP1, and SREBP. Kinases and other signaling proteins are perfectly suited to regulate life and death decisions in response to cellular stressors and have only recently been identified as important caspase substrates. Here we review the current status of signaling pathways that are activated, inactivated or dysregulated by proteases such as caspases and calpain to control entry into apoptosis. The emerging concept that some caspase pathways may be inhibited by cellular and viral apoptosis inhibitory proteins while other caspase pathways are preserved suggests that a subset of these kinases may exist as cleaved 'isoforms' in cells that are not destined to perish. By acting as executioners and as important 'molecular sensors' of the degree of cellular injury, the signaling proteins described in this review are strong candidates to mediate downstream events, both in condemned and in viable cells.
View details for Web of Science ID 000088066000002
View details for PubMedID 10889504
The fate of U1 snRNP during anti-Fas induced apoptosis: specific cleavage of the U1 snRNA molecule
CELL DEATH AND DIFFERENTIATION
2000; 7 (1): 70-79
During apoptosis, the U1-70K protein, a component of the spliceosomal U1 snRNP complex, is specifically cleaved by the enzyme caspase-3, converting it into a C-terminally truncated 40-kDa fragment. In this study, we show that the 40-kDa U1-70K fragment is still associated with the complete U1 snRNP complex, and that no obvious modifications occur with the U1 snRNP associated proteins U1A, U1C and Sm-B/B'. Furthermore, it is described for the first time that the U1 snRNA molecule, which is the backbone of the U1 snRNP complex, is modified during apoptosis by the specific removal of the first 5 - 6 nucleotides including the 2,2, 7-trimethylguanosine (TMG) cap. The observations that U1 snRNA cleavage is very specific (no such modifications were detected for the other U snRNAs tested) and that U1 snRNA cleavage is markedly inhibited in the presence of caspase inhibitors, indicate that an apoptotically activated ribonuclease is responsible for the specific modification of U1 snRNA during apoptosis.
View details for Web of Science ID 000085885600009
View details for PubMedID 10713722
The La (SS-B) autoantigen, a key protein in RNA biogenesis, is dephosphorylated and cleaved early during apoptosis
CELL DEATH AND DIFFERENTIATION
1999; 6 (10): 976-986
In the past few years, a role for apoptotic processes in the development of autoimmune diseases has been suggested. An increasing number of cellular proteins, which are modified during apoptosis, has been described, and many of these proteins have been identified as autoantigens. We have studied the effects of apoptosis on the La protein in more detail and for the first time demonstrate that this autoantigen is rapidly dephosphorylated after the induction of apoptosis. Dephosphorylation of the La protein was observed after induction of apoptosis by several initiators and in various cell types. Furthermore, we demonstrate that at least a subset of the La protein is proteolytically cleaved in vivo, generating a 45 kDa fragment. Dephosphorylation as well as cleavage of La is inhibited by ZnSO4 as well as by several tetrapeptide caspase inhibitors, indicating that these processes require the activation of caspases. Dephosphorylation of La is inhibited by low concentrations of okadaic acid, suggesting that a PP2A-like phosphatase is involved. Generation of the 45 kDa fragment is consistent with proteolytic cleavage at amino acids 371 and/or 374. The possible significance of the apoptotic changes in the La protein for autoantibody production is discussed.
View details for Web of Science ID 000083259800009
View details for PubMedID 10556975
Rapid nucleolytic degradation of the small cytoplasmic Y RNAs during apoptosis
JOURNAL OF BIOLOGICAL CHEMISTRY
1999; 274 (35): 24799-24807
We have investigated the fate of the RNA components of small ribonucleoprotein particles in apoptotic cells. We show that the cytoplasmic Ro ribonucleoprotein-associated Y RNAs are specifically and rapidly degraded during apoptosis via a caspase-dependent mechanism. This is the first study describing the selective degradation of a specific class of small structural RNA molecules in apoptotic cells. Cleavage and subsequent truncation of Y RNAs was observed upon exposure of cells to a variety of apoptotic stimuli and were found to be inhibited by Bcl-2, zinc, and several caspase inhibitors. These results indicate that apoptotic degradation of Y RNAs is dependent on caspase activation, which suggests that the nucleolytic activity responsible for hY RNA degradation is activated downstream of the caspase cascade. The Y RNA degradation products remain bound by the Ro60 protein and in part also by the La protein, the only two proteins known to be stably associated with intact Ro ribonucleoprotein particles. The size of the Y RNA degradation products is consistent with the protection from degradation of the most highly conserved region of the Y RNAs by the bound Ro60 and La proteins. Our results indicate that the rapid abrogation of the yet unknown function of Y RNAs might be an early step in the systemic deactivation of the dying cell.
View details for Web of Science ID 000082193400051
View details for PubMedID 10455152
SUP-HD1 - A NEW HODGKINS DISEASE-DERIVED CELL-LINE WITH LYMPHOID FEATURES PRODUCES INTERFERON-GAMMA
1989; 74 (8): 2733-2742
A new cell line, SUP-HD1, was established from the pleural effusion of a patient with nodular sclerosing Hodgkin's disease (NSHD). The SUP-HD1 cells had the characteristic morphology of Reed-Sternberg cells and contained acid phosphatase and nonspecific esterase. The cells lacked the Epstein-Barr virus (EBV) genome and reacted with monoclonal antibodies (MoAbs) against CD15 (Leu-M1), CD25 (Tac), CD71 (OKT9), Ki67, and HLA-Dr. However, the SUP-HD1 cells were nonreactive with MoAbs that specifically identify T lymphocytes, B lymphocytes, and macrophage/myeloid cells. Karyotype analysis of the cell line showed clonal abnormalities involving 1p13, 7p15, 8q22, and 11q23, chromosomal locations, at which breakpoints have been reported in HD. Southern blot analysis demonstrated rearrangement of the immunoglobulin heavy chain and kappa light chain genes as well as the gene for the beta chain of the T-cell receptor (TCR). Transcriptional analysis showed expression of RNAs for kappa light chain, interferon-gamma (IFN-gamma), and interleukin-2 receptor (IL-2R) but not IL-2. The SUP-HD1 cells lacked cytoplasmic and surface immunoglobulin heavy chain, but a small amount of cytoplasmic kappa light chain was detected. The presence of nuclear factor kappa B (NF kappa B), a B-lymphocyte-associated transcription factor, was demonstrated in stimulated and unstimulated cells. In addition, the SUP-HD1 cell line, produced IFN-gamma, a T-lymphocyte-associated lymphokine. Based on these data, the SUP-HD1 cells appear to be aberrant lymphocytes with characteristics of both activated B and T lymphocytes. Elaboration of lymphokines such as IFN-gamma by the malignant cells may represent one explanation for the unique clinical and pathologic features of HD.
View details for Web of Science ID A1989CB88100019
View details for PubMedID 2554995