PJ Utz

All Publications

• Mycophenolate mofetil reduces STAT3 phosphorylation in systemic lupus erythematosus patients. JCI insight Slight-Webb, S., Guthridge, J. M., Chakravarty, E. F., Chen, H., Lu, R., Macwana, S., Bean, K., Maecker, H. T., Utz, P. J., James, J. A. 2019; 4 (2)

  Abstract

  Systemic lupus erythematosus (SLE) is a highly variable autoimmune disease that can involve severe organ-threatening symptoms, such as lupus nephritis. Certain drugs, such as mycophenolate mofetil (MMF), are effective at reducing morbidity associated with nephritis; however, the immune pathways associated with disease suppression are poorly defined. Here, we provide evidence that MMF inhibits phosphorylation of STAT3 and other associated immune pathways. Using mass cytometry and bead-based or ELISA assays, the systemic phenotype of SLE patients not taking (MMF-) or taking (MMF+) MMF were studied. MMF+ SLE patients had significant reductions in total numbers of transitional B cells, plasmablasts, and T cells, specifically CD4+ Th17-type and CD4+ Treg-type cells, compared with MMF- patients. Plasma soluble mediators were decreased in MMF+ patients including chemokines (MIG/CXCL9 and SDF-1alpha/CXCL12) and growth factors (VEGF-A and PDGF-BB). Soluble mediators and cell subsets grouped by functional properties revealed significant modifications associated with STAT3 and B cell pathways. Further, healthy PBMCs treated with IL-6 revealed a reduction in p-STAT3 following the addition of mycophenolic acid (the active metabolite of MMF). In conclusion, the inhibition of STAT3 phosphorylation by MMF may explain the effectiveness of this treatment in SLE patients, since increased levels of p-STAT3 are associated with disease pathology.

  View details for DOI 10.1172/jci.insight.124575

  View details for PubMedID 30674728

• Quantification of cDNA on GMR biosensor array towards point-of-care gene expression analysis. Biosensors & bioelectronics Ravi, N., Rizzi, G., Chang, S. E., Cheung, P., Utz, P. J., Wang, S. X. 2018

  Abstract

  Gene expression analysis at the point-of-care is important for rapid disease diagnosis, but traditional techniques are limited by multiplexing capabilities, bulky equipment, and cost. We present a gene expression analysis platform using a giant magnetoresistive (GMR) biosensor array, which allows multiplexed transcript detection and quantification through cost-effective magnetic detection. In this work, we have characterized the sensitivity, dynamic range, and quantification accuracy of Polymerase chain reaction (PCR)-amplified complementary DNA (cDNA) on the GMR for the reference gene GAPDH. A synthetic GAPDH single-stranded DNA (ssDNA) standard was used to calibrate the detection, and ssDNA dilutions were qPCR-amplified to obtain a standard curve. We demonstrate that the GMR platform provides a dynamic range of 4 orders of magnitude and a limit of detection of 1 pM and 0.1 pM respectively for 15 and 18-cycle amplified synthetic GAPDH PCR products. The quantitative results of GMR analysis of cell-line RNA were confirmed by qPCR.

  View details for DOI 10.1016/j.bios.2018.09.050

  View details for PubMedID 30269961

• African American and European American SLE Patients with Variable Disease Activity Reveal Distinct Differences in B Cells and TLR7/8 Pathways Slight-Webb, S., Smith, M. C., Maecker, H. T., Utz, P. J., Guthridge, J. M., James, J. A. WILEY. 2018

  View details for Web of Science ID 000447268905271

• Multiplexed Autoantibody Profiles in a Systemic Sclerosis Clinical Trial Comparing Autologous Hematopoietic Stem Cell Transplantation and Cyclophosphamide Utz, P. J., Ayoglu, B. WILEY. 2018

  View details for Web of Science ID 000447268902836

• Diminished STAT-3 Phosphorylation and Associated Cell Pathways Characterize MMF-Treated Systemic Lupus Erythematosus Patients Slight-Webb, S., Guthridge, J. M., Chakravarty, E., Chen, H., Lu, R., Bean, K. M., Maecker, H. T., Utz, P. J., James, J. A. WILEY. 2018

  View details for Web of Science ID 000447268905159

• Pro-Inflammatory and Regulatory Soluble Mediator Pathways Vary between African American and European American SLE Patients Slight-Webb, S., Smith, M. C., Maecker, H. T., Utz, P. J., Guthridge, J. M., James, J. A. WILEY. 2018

  View details for Web of Science ID 000447268902189

• Methods for high-dimensonal analysis of cells dissociated from cyropreserved synovial tissue ARTHRITIS RESEARCH & THERAPY Donlin, L. T., Rao, D. A., Wei, K., Slowikowski, K., McGeachy, M. J., Turner, J. D., Meednu, N., Mizoguchi, F., Gutierrez-Arcelus, M., Lieb, D. J., Keegan, J., Muskat, K., Hillman, J., Rozo, C., Ricker, E., Eisenhaure, T. M., Li, S., Browne, E. P., Chicoine, A., Sutherby, D., Noma, A., Nusbaum, C., Kelly, S., Pernis, A. B., Ivashkiv, L. B., Goodman, S. M., Robinson, W. H., Utz, P. J., Lederer, J. A., Gravallese, E. M., Boyce, B. F., Hacohen, N., Pitzalis, C., Gregersen, P. K., Firestein, G. S., Raychaudhuri, S., Moreland, L. W., Holers, V., Bykerk, V. P., Filer, A., Boyle, D. L., Brenner, M. B., Anolik, J. H., Accelerating Med Partnership RA SL 2018; 20: 139

  Abstract

  Detailed molecular analyses of cells from rheumatoid arthritis (RA) synovium hold promise in identifying cellular phenotypes that drive tissue pathology and joint damage. The Accelerating Medicines Partnership RA/SLE Network aims to deconstruct autoimmune pathology by examining cells within target tissues through multiple high-dimensional assays. Robust standardized protocols need to be developed before cellular phenotypes at a single cell level can be effectively compared across patient samples.Multiple clinical sites collected cryopreserved synovial tissue fragments from arthroplasty and synovial biopsy in a 10% DMSO solution. Mechanical and enzymatic dissociation parameters were optimized for viable cell extraction and surface protein preservation for cell sorting and mass cytometry, as well as for reproducibility in RNA sequencing (RNA-seq). Cryopreserved synovial samples were collectively analyzed at a central processing site by a custom-designed and validated 35-marker mass cytometry panel. In parallel, each sample was flow sorted into fibroblast, T-cell, B-cell, and macrophage suspensions for bulk population RNA-seq and plate-based single-cell CEL-Seq2 RNA-seq.Upon dissociation, cryopreserved synovial tissue fragments yielded a high frequency of viable cells, comparable to samples undergoing immediate processing. Optimization of synovial tissue dissociation across six clinical collection sites with ~ 30 arthroplasty and ~ 20 biopsy samples yielded a consensus digestion protocol using 100 μg/ml of Liberase™ TL enzyme preparation. This protocol yielded immune and stromal cell lineages with preserved surface markers and minimized variability across replicate RNA-seq transcriptomes. Mass cytometry analysis of cells from cryopreserved synovium distinguished diverse fibroblast phenotypes, distinct populations of memory B cells and antibody-secreting cells, and multiple CD4+ and CD8+ T-cell activation states. Bulk RNA-seq of sorted cell populations demonstrated robust separation of synovial lymphocytes, fibroblasts, and macrophages. Single-cell RNA-seq produced transcriptomes of over 1000 genes/cell, including transcripts encoding characteristic lineage markers identified.We have established a robust protocol to acquire viable cells from cryopreserved synovial tissue with intact transcriptomes and cell surface phenotypes. A centralized pipeline to generate multiple high-dimensional analyses of synovial tissue samples collected across a collaborative network was developed. Integrated analysis of such datasets from large patient cohorts may help define molecular heterogeneity within RA pathology and identify new therapeutic targets and biomarkers.

  View details for DOI 10.1186/s13075-018-1631-y

  View details for Web of Science ID 000438396800002

  View details for PubMedID 29996944

  View details for PubMedCentralID PMC6042350

• Single-cell epigenetics - Chromatin modification atlas unveiled by mass cytometry. Clinical immunology (Orlando, Fla.) Cheung, P., Vallania, F., Dvorak, M., Chang, S. E., Schaffert, S., Donato, M., Rao, A., Mao, R., Utz, P. J., Khatri, P., Kuo, A. J. 2018

  Abstract

  Modifications of histone proteins are fundamental to the regulation of epigenetic phenotypes. Dysregulations of histone modifications have been linked to the pathogenesis of diverse human diseases. However, identifying differential histone modifications in patients with immune-mediated diseases has been challenging, in part due to the lack of a powerful analytic platform to study histone modifications in the complex human immune system. We recently developed a highly multiplexed platform, Epigenetic landscape profiling using cytometry by Time-Of-Flight (EpiTOF), to analyze the global levels of a broad array of histone modifications in single cells using mass cytometry. In this review, we summarize the development of EpiTOF and discuss its potential applications in biomedical research. We anticipate that this platform will provide new insights into the roles of epigenetic regulation in hematopoiesis, immune cell functions and immune system aging, and reveal aberrant epigenetic patterns associated with immune-mediated diseases.

  View details for DOI 10.1016/j.clim.2018.06.009

  View details for PubMedID 29960011

• KLRD1-expressing natural killer cells predict influenza susceptibility GENOME MEDICINE Bongen, E., Vallania, F., Utz, P. J., Khatri, P. 2018; 10: 45

  Abstract

  Influenza infects tens of millions of people every year in the USA. Other than notable risk groups, such as children and the elderly, it is difficult to predict what subpopulations are at higher risk of infection. Viral challenge studies, where healthy human volunteers are inoculated with live influenza virus, provide a unique opportunity to study infection susceptibility. Biomarkers predicting influenza susceptibility would be useful for identifying risk groups and designing vaccines.We applied cell mixture deconvolution to estimate immune cell proportions from whole blood transcriptome data in four independent influenza challenge studies. We compared immune cell proportions in the blood between symptomatic shedders and asymptomatic nonshedders across three discovery cohorts prior to influenza inoculation and tested results in a held-out validation challenge cohort.Natural killer (NK) cells were significantly lower in symptomatic shedders at baseline in both discovery and validation cohorts. Hematopoietic stem and progenitor cells (HSPCs) were higher in symptomatic shedders at baseline in discovery cohorts. Although the HSPCs were higher in symptomatic shedders in the validation cohort, the increase was statistically nonsignificant. We observed that a gene associated with NK cells, KLRD1, which encodes CD94, was expressed at lower levels in symptomatic shedders at baseline in discovery and validation cohorts. KLRD1 expression in the blood at baseline negatively correlated with influenza infection symptom severity. KLRD1 expression 8 h post-infection in the nasal epithelium from a rhinovirus challenge study also negatively correlated with symptom severity.We identified KLRD1-expressing NK cells as a potential biomarker for influenza susceptibility. Expression of KLRD1 was inversely correlated with symptom severity. Our results support a model where an early response by KLRD1-expressing NK cells may control influenza infection.

  View details for DOI 10.1186/s13073-018-0554-1

  View details for Web of Science ID 000435421500001

  View details for PubMedID 29898768

  View details for PubMedCentralID PMC6001128

• Single-Cell Chromatin Modification Profiling Reveals Increased Epigenetic Variations with Aging. Cell Cheung, P., Vallania, F., Warsinske, H. C., Donato, M., Schaffert, S., Chang, S. E., Dvorak, M., Dekker, C. L., Davis, M. M., Utz, P. J., Khatri, P., Kuo, A. J. 2018

  Abstract

  Post-translational modifications of histone proteins and exchanges of histone variants of chromatin are central to the regulation of nearly all DNA-templated biological processes. However, the degree and variability of chromatin modifications in specific human immune cells remain largely unknown. Here, we employ a highly multiplexed mass cytometry analysis to profile the global levels of a broad array of chromatin modifications in primary human immune cells at the single-cell level. Our data reveal markedly different cell-type- and hematopoietic-lineage-specific chromatin modification patterns. Differential analysis between younger and older adults shows that aging is associated with increased heterogeneity between individuals and elevated cell-to-cell variability in chromatin modifications. Analysis of a twin cohort unveils heritability of chromatin modifications and demonstrates that aging-related chromatin alterations are predominantly driven by non-heritable influences. Together, we present a powerful platform for chromatin and immunology research. Our discoveries highlight the profound impacts of aging on chromatin modifications.

  View details for DOI 10.1016/j.cell.2018.03.079

  View details for PubMedID 29706550

• Neutralizing Anti-Cytokine Autoantibodies Against Interferon-alpha in Immunodysregulation Polyendocrinopathy Enteropathy X-Linked FRONTIERS IN IMMUNOLOGY Rosenberg, J. M., Maccari, M. E., Barzaghi, F., Allenspach, E. J., Pignata, C., Weber, G., Torgerson, T. R., Utz, P. J., Bacchetta, R. 2018; 9: 544

  Abstract

  Anti-cytokine autoantibodies (ACAAs) have been described in a growing number of primary immunodeficiencies with autoimmune features, including autoimmune polyendocrine syndrome type I (APS-1), a prototypical disease of defective T cell-mediated central tolerance. Whether defects in peripheral tolerance lead to similar ACAAs is unknown. Immunodysregulation polyendocrinopathy enteropathy X-linked (IPEX) is caused by mutations in FOXP3, a master regulator of T regulatory cells (Treg), and consequently results in defective T cell-mediated peripheral tolerance. Unique autoantibodies have previously been described in IPEX. To test the hypothesis that ACAAs are present in IPEX, we designed and fabricated antigen microarrays. We discovered elevated levels of IgG ACAAs against interferon-α (IFN-α) in a cohort of IPEX patients. Serum from IPEX patients blocked IFN-α signaling in vitro and blocking activity was tightly correlated with ACAA titer. To show that blocking activity was mediated by IgG and not other serum factors, we purified IgG and showed that blocking activity was contained entirely in the immunoglobulin fraction. We also screened for ACAAs against IFN-α in a second geographically distinct cohort. In these samples, ACAAs against IFN-α were elevated in a post hoc analysis. In summary, we report the discovery of ACAAs against IFN-α in IPEX, an experiment of nature demonstrating the important role of peripheral T cell tolerance.

  View details for DOI 10.3389/fimmu.2018.00544

  View details for Web of Science ID 000428633300001

  View details for PubMedID 29651287

  View details for PubMedCentralID PMC5885158

• Using Flow and Mass Cytometry to Demonstrate Robust Tissue Processing to Query Molecular Heterogeneity in Phase 1 of the Accelerating Medicines Partnership (AMP) - RA Network Wei, K., Rao, D., Zhang, F., Fonseka, C., Slowikowski, K., Keegan, J., Donlin, L. T., Turner, J., McGeachy, M. J., Meednu, N., Lieb, D., Kelly, S., Goodman, S. M., Boyle, D. L., Robinson, W. H., Utz, P. J., Firestein, G. S., Perlman, H., DiCarlo, E. F., Pitzalis, C., Filer, A., Boyce, B., Gravallese, E. M., Nusbaum, C., Lederer, J., Hacohen, N., Gregersen, P., Moreland, L. W., Holers, M., Bykerk, V. P., Raychaudhuri, S., Brenner, M., Anolik, J. H. WILEY. 2017

  View details for Web of Science ID 000411824102322

• Identification of Biomarkers Predictive of Pulmonary Arterial Hypertension in Systemic Sclerosis Kolstad, K. D., Holmes, T., Rosenberg-Hasson, Y., Sweatt, A., Zamanian, R. T., Li, S., Steen, V. D., Utz, P. J., Chung, L. WILEY. 2017

  View details for Web of Science ID 000411824101041

• Whole Blood Stimulations Identify Elevated T Cell Cytokines and Altered Granulocyte/Dendritic Cell Signaling in SLE Patients with Variable Disease Activity Slight-Webb, S., Bean, K. M., Maecker, H. T., Utz, P. J., James, J. A., Guthridge, J. M. WILEY. 2017

  View details for Web of Science ID 000411824100084

• Molecular Features Define Unique Sjogren's Syndrome Patient Subsets James, J. A., Guthridge, J. M., Chen, H., Lu, R., Bourn, R. L., Baer, A. N., Noaiseh, G., Parke, A., Coca, A., Utset, T., Genovese, M. C., Aberle, T., Wallace, D. J., Boyle, K., Keyes-Elstein, L., Franchimont, N., Clair, E., Pascual, V., Utz, P. J., Sivils, K. L. WILEY. 2017

  View details for Web of Science ID 000411824106379

• Lupus Nephritis in Isolation or Accompanied By Extra-Renal Manifestations: Early Lessons from the Accelerating Medicines Partnership James, J. A., Petri, M., Putterman, C., Diamond, B., Wofsy, D., Lee, C., Fine, D., Broder, A. R., Clancy, R. M., Izmirly, P. M., Belmont, M., Bornkamp, N., Davidson, A., Tosta, P., Kalunian, K. C., Park, M., Dall'Era, M., Furie, R., Massarotti, E., Hernandez, G. T., Payan-Schober, F., Connery, S. M., Kamen, D. L., Lee, I., Pendergraft, W., Anolik, J. H., Shah, U., Raychaudhuri, S., Lee, Y. C., Guthridge, J. M., Holers, V., Utz, P. J., Pichavant, M., Gupta, R., Maecker, H. T., Weisman, M., Buyon, J. P. WILEY. 2017

  View details for Web of Science ID 000411824101002

• Severe Toxoplasma gondii infection in a member of a NFKB2-deficient family with T and B cell dysfunction CLINICAL IMMUNOLOGY Maccari, M., Scarselli, A., Di Cesare, S., Floris, M., Angius, A., Deodati, A., Chiriaco, M., Cambiaso, P., Corrente, S., Colafati, G., Utz, P. J., Angelini, F., Fierabracci, A., Aiuti, A., Carsetti, R., Rosenberg, J. M., Cappa, M., Rossi, P., Bacchetta, R., Cancrini, C. 2017; 183: 273–77

  View details for DOI 10.1016/j.clim.2017.09.011

  View details for Web of Science ID 000414888600037

  View details for PubMedID 28919517

• Hydroxychloroquine Use Is Associated with Decreased Dendritic Cell Activation and Thrombolytic Factors in SLE Patients Slight-Webb, S., Lu, R., Chen, H., Maecker, H. T., Utz, P. J., Guthridge, J. M., James, J. A. WILEY. 2017

  View details for Web of Science ID 000411824106100

• Smith-Magenis Syndrome Patients Often Display Antibody Deficiency but Not Other Immune Pathologies JOURNAL OF ALLERGY AND CLINICAL IMMUNOLOGY-IN PRACTICE Perkins, T., Rosenberg, J. M., Le Coz, C., Alaimo, J. T., Trofa, M., Mullegama, S. V., Antaya, R. J., Jyonouchi, S., Elsea, S. H., Utz, P. J., Meffre, E., Romberg, N. 2017; 5 (5): 1344-+

  Abstract

  Smith-Magenis syndrome (SMS) is a complex neurobehavioral disorder associated with recurrent otitis. Most SMS cases result from heterozygous interstitial chromosome 17p11.2 deletions that encompass not only the intellectual disability gene retinoic acid-induced 1 but also other genes associated with immunodeficiency, autoimmunity, and/or malignancy.The goals of this study were to describe the immunological consequence of 17p11.2 deletions by determining the prevalence of immunological diseases in subjects with SMS and by assessing their immune systems via laboratory methods.We assessed clinical histories of 76 subjects with SMS with heterozygous 17p11.2 deletions and performed in-depth immunological testing on 25 representative cohort members. Laboratory testing included determination of serum antibody concentrations, vaccine titers, and lymphocyte subset frequencies. Detailed reactivity profiles of SMS serum antibodies were performed using custom-made antigen microarrays.Of 76 subjects with SMS, 74 reported recurrent infections including otitis (88%), pneumonia (47%), sinusitis (42%), and gastroenteritis (34%). Infections were associated with worsening SMS-related neurobehavioral symptoms. The prevalence of autoimmune and atopic diseases was not increased. Malignancy was not reported. Laboratory evaluation revealed most subjects with SMS to be deficient of isotype-switched memory B cells and many to lack protective antipneumococcal antibodies. SMS antibodies were not more reactive than control antibodies to self-antigens.Patients with SMS with heterozygous 17p.11.2 deletions display an increased susceptibility to sinopulmonary infections, but not to autoimmune, allergic, or malignant diseases. SMS sera display an antibody reactivity profile favoring neither recognition of pathogen-associated antigens nor self-antigens. Prophylactic strategies to prevent infections may also provide neurobehavioral benefits to selected patients with SMS.

  View details for DOI 10.1016/j.jaip.2017.01.028

  View details for Web of Science ID 000409364200024

  View details for PubMedID 28286158

  View details for PubMedCentralID PMC5591748

• Proteomic Analysis of Sera from Individuals with Diffuse Cutaneous Systemic Sclerosis Reveals a Multianalyte Signature Associated with Clinical Improvement during Imatinib Mesylate Treatment JOURNAL OF RHEUMATOLOGY Haddon, D. J., Wand, H. E., Jarrell, J. A., Spiera, R. F., Utz, P. J., Gordon, J. K., Chung, L. S. 2017; 44 (5): 631-638

  Abstract

  Imatinib has been investigated for the treatment of systemic sclerosis (SSc) because of its ability to inhibit the platelet-derived growth factor receptor and transforming growth factor-β signaling pathways, which have been implicated in SSc pathogenesis. In a 12-month open-label clinical trial assessing the safety and efficacy of imatinib in the treatment of diffuse cutaneous SSc (dcSSc), significant improvements in skin thickening were observed. Here, we report our analysis of sera collected during the clinical trial.We measured the levels of 46 cytokines, chemokines, and growth factors in the sera of individuals with dcSSc using Luminex and ELISA. Autoantigen microarrays were used to measure immunoglobulin G reactivity to 28 autoantigens. Elastic net regularization was used to identify a signature that was predictive of clinical improvement (reduction in the modified Rodnan skin score ≥ 5) during treatment with imatinib. The signature was also tested using sera from a clinical trial of nilotinib, a tyrosine kinase inhibitor that is structurally related to imatinib, in dcSSc.The elastic net algorithm identified a signature, based on levels of CD40 ligand, chemokine (C-X-C motif) ligand 4 (CXCL4), and anti-PM/Scl-100, that was significantly higher in individuals who experienced clinical improvement than in those who did not (p = 0.0011). The signature was validated using samples from a clinical trial of nilotinib.Identification of patients with SSc with the greatest probability of benefit from treatment with imatinib has the potential to guide individualized treatment. Validation of the signature will require testing in randomized, placebo-controlled studies. Clinicaltrials.gov NCT00555581 and NCT01166139.

  View details for DOI 10.3899/jrheum.160833

  View details for Web of Science ID 000401094300016

  View details for PubMedID 28298564

• Clonal Evolution of Autoreactive Germinal Centers. Cell Degn, S. E., van der Poel, C. E., Firl, D. J., Ayoglu, B., Al Qureshah, F. A., Bajic, G., Mesin, L., Reynaud, C. A., Weill, J. C., Utz, P. J., Victora, G. D., Carroll, M. C. 2017; 170 (5): 913–26.e19

  Abstract

  Germinal centers (GCs) are the primary sites of clonal B cell expansion and affinity maturation, directing the production of high-affinity antibodies. This response is a central driver of pathogenesis in autoimmune diseases, such as systemic lupus erythematosus (SLE), but the natural history of autoreactive GCs remains unclear. Here, we present a novel mouse model where the presence of a single autoreactive B cell clone drives the TLR7-dependent activation, expansion, and differentiation of other autoreactive B cells in spontaneous GCs. Once tolerance was broken for one self-antigen, autoreactive GCs generated B cells targeting other self-antigens. GCs became independent of the initial clone and evolved toward dominance of individual clonal lineages, indicating affinity maturation. This process produced serum autoantibodies to a breadth of self-antigens, leading to antibody deposition in the kidneys. Our data provide insight into the maturation of the self-reactive B cell response, contextualizing the epitope spreading observed in autoimmune disease.

  View details for DOI 10.1016/j.cell.2017.07.026

  View details for PubMedID 28841417

• Integrated, multicohort analysis of systemic sclerosis identifies robust transcriptional signature of disease severity. JCI insight Lofgren, S., Hinchcliff, M., Carns, M., Wood, T., Aren, K., Arroyo, E., Cheung, P., Kuo, A., Valenzuela, A., Haemel, A., Wolters, P. J., Gordon, J., Spiera, R., Assassi, S., Boin, F., Chung, L., Fiorentino, D., Utz, P. J., Whitfield, M. L., Khatri, P. 2016; 1 (21)

  Abstract

  Systemic sclerosis (SSc) is a rare autoimmune disease with the highest case-fatality rate of all connective tissue diseases. Current efforts to determine patient response to a given treatment using the modified Rodnan skin score (mRSS) are complicated by interclinician variability, confounding, and the time required between sequential mRSS measurements to observe meaningful change. There is an unmet critical need for an objective metric of SSc disease severity. Here, we performed an integrated, multicohort analysis of SSc transcriptome data across 7 datasets from 6 centers composed of 515 samples. Using 158 skin samples from SSc patients and healthy controls recruited at 2 centers as a discovery cohort, we identified a 415-gene expression signature specific for SSc, and validated its ability to distinguish SSc patients from healthy controls in an additional 357 skin samples from 5 independent cohorts. Next, we defined the SSc skin severity score (4S). In every SSc cohort of skin biopsy samples analyzed in our study, 4S correlated significantly with mRSS, allowing objective quantification of SSc disease severity. Using transcriptome data from the largest longitudinal trial of SSc patients to date, we showed that 4S allowed us to objectively monitor individual SSc patients over time, as (a) the change in 4S of a patient is significantly correlated with change in the mRSS, and (b) the change in 4S at 12 months of treatment could predict the change in mRSS at 24 months. Our results suggest that 4S could be used to distinguish treatment responders from nonresponders prior to mRSS change. Our results demonstrate the potential clinical utility of a novel robust molecular signature and a computational approach to SSc disease severity quantification.

  View details for DOI 10.1172/jci.insight.89073

  View details for PubMedID 28018971

  View details for PubMedCentralID PMC5161207

• High-Resolution Analysis of Antibodies to Post-Translational Modifications Using Peptide Nanosensor Microarrays ACS NANO Lee, J., Haddon, D. J., Gupta, N., Price, J. V., Credo, G. M., Diep, V. K., Kim, K., Hall, D. A., Baechler, E. C., Petri, M., Varma, M., Utz, P. J., Wang, S. X. 2016; 10 (12): 10652-10660

  Abstract

  Autoantibodies are a hallmark of autoimmune diseases such as lupus and have the potential to be used as biomarkers for diverse diseases, including immunodeficiency, infectious disease, and cancer. More precise detection of antibodies to specific targets is needed to improve diagnosis of such diseases. Here, we report the development of reusable peptide microarrays, based on giant magnetoresistive (GMR) nanosensors optimized for sensitively detecting magnetic nanoparticle labels, for the detection of antibodies with a resolution of a single post-translationally modified amino acid. We have also developed a chemical regeneration scheme to perform multiplex assays with a high level of reproducibility, resulting in greatly reduced experimental costs. In addition, we show that peptides synthesized directly on the nanosensors are approximately two times more sensitive than directly spotted peptides. Reusable peptide nanosensor microarrays enable precise detection of autoantibodies with high resolution and sensitivity and show promise for investigating antibody-mediated immune responses to autoantigens, vaccines, and pathogen-derived antigens as well as other fundamental peptide-protein interactions.

  View details for DOI 10.1021/acsnano.6b03786

  View details for Web of Science ID 000391079700007

  View details for PubMedID 27636738

• Portable, one-step, and rapid GMR biosensor platform with smartphone interface. Biosensors & bioelectronics Choi, J., Gani, A. W., Bechstein, D. J., Lee, J., Utz, P. J., Wang, S. X. 2016; 85: 1-7

  Abstract

  Quantitative immunoassay tests in clinical laboratories require trained technicians, take hours to complete with multiple steps, and the instruments used are generally immobile-patient samples have to be sent in to the labs for analysis. This prevents quantitative immunoassay tests to be performed outside laboratory settings. A portable, quantitative immunoassay device will be valuable in rural and resource-limited areas, where access to healthcare is scarce or far away. We have invented Eigen Diagnosis Platform (EDP), a portable quantitative immunoassay platform based on Giant Magnetoresistance (GMR) biosensor technology. The platform does not require a trained technician to operate, and only requires one-step user involvement. It displays quantitative results in less than 15min after sample insertion, and each test costs less than US$4. The GMR biosensor employed in EDP is capable of detecting multiple biomarkers in one test, enabling a wide array of immune diagnostics to be performed simultaneously. In this paper, we describe the design of EDP, and demonstrate its capability. Multiplexed assay of human immunoglobulin G and M (IgG and IgM) antibodies with EDP achieves sensitivities down to 0.07 and 0.33 nanomolar, respectively. The platform will allow lab testing to be performed in remote areas, and open up applications of immunoassay testing in other non-clinical settings, such as home, school, and office.

  View details for DOI 10.1016/j.bios.2016.04.046

  View details for PubMedID 27148826

• Broad-spectrum antibodies against self-antigens and cytokines in RAG deficiency. journal of clinical investigation Walter, J. E., Rosen, L. B., Csomos, K., Rosenberg, J. M., Mathew, D., Keszei, M., Ujhazi, B., Chen, K., Lee, Y. N., Tirosh, I., Dobbs, K., Al-Herz, W., Cowan, M. J., Puck, J., Bleesing, J. J., Grimley, M. S., Malech, H., De Ravin, S. S., Gennery, A. R., Abraham, R. S., Joshi, A. Y., Boyce, T. G., Butte, M. J., Nadeau, K. C., Balboni, I., Sullivan, K. E., Akhter, J., Adeli, M., El-Feky, R. A., El-Ghoneimy, D. H., Dbaibo, G., Wakim, R., Azzari, C., Palma, P., Cancrini, C., Capuder, K., Condino-Neto, A., Costa-Carvalho, B. T., Oliveira, J. B., Roifman, C., Buchbinder, D., Kumanovics, A., Franco, J. L., Niehues, T., Schuetz, C., Kuijpers, T., Yee, C., Chou, J., Masaad, M. J., Geha, R., Uzel, G., Gelman, R., Holland, S. M., Recher, M., Utz, P. J., Browne, S. K., Notarangelo, L. D. 2016; 126 (11): 4389-?

  View details for DOI 10.1172/JCI91162

  View details for PubMedID 27801680

  View details for PubMedCentralID PMC5096894

• Mycophenolate Mofetil Use Associates with Unique Biologic Changes in B Cell and T Regulatory Cell Pathways in SLE Patients Slight-Webb, S., Lu, R., Bean, K. M., Maecker, H. T., Utz, P. J., Guthridge, J. M., James, J. A. WILEY. 2016

  View details for Web of Science ID 000417143403004

• Autoantibody-Positive Healthy Individuals Display Unique Immune Profiles That May Regulate Autoimmunity. Arthritis & rheumatology Slight-Webb, S., Lu, R., Ritterhouse, L. L., Munroe, M. E., Maecker, H. T., Fathman, C. G., Utz, P. J., Merrill, J. T., Guthridge, J. M., James, J. A. 2016; 68 (10): 2492-2502

  Abstract

  Antinuclear antibodies (ANAs) are detected in ∼18% of females, yet autoimmune disease develops in only 5-8%. Immunologic differences between ANA-positive healthy individuals and patients with systemic lupus erythematosus (SLE) may elucidate the regulatory mechanisms by which ANA-positive individuals avoid transition to clinical autoimmune disease.Healthy individuals (n = 790) were screened for autoantibodies specific for 11 antigens associated with lupus, systemic sclerosis, and Sjögren's syndrome. From this screening, 31 European American ANA-positive healthy individuals were selected and demographically matched to ANA-negative controls and SLE patients. Serum cytokine profiles, leukocyte subset frequency, and reactivity were analyzed by multiplex assays, immunophenotyping, and phosphospecific flow cytometry.Of 790 individuals screened, 57 (7%) were ANA-positive. The majority of proinflammatory cytokines, including interferon-γ (IFNγ), tumor necrosis factor, interleukin-17 (IL-17), and granulocyte colony-stimulating factor, exhibited a stepwise increase in serum levels from ANA-negative controls to ANA-positive healthy individuals to SLE patients (P < 0.0001). IFNα, IFNβ, IL-12p40, and stem cell factor/c-Kit ligand were increased in SLE patients only (P < 0.05). B lymphocyte stimulator (BlyS) was elevated in SLE patients but decreased in ANA-positive individuals (P < 0.001). Further, IL-1 receptor antagonist (IL-1Ra) was down-regulated in SLE patients only (P < 0.0001). ANA-positive individuals had increased frequencies of monocytes, memory B cells, and plasmablasts and increased levels of pSTAT-1 and pSTAT-3 following IFNα stimulation compared with ANA-negative controls (P < 0.05).ANA-positive healthy individuals exhibit dysregulation in multiple immune pathways yet differ from SLE patients by the absence of elevated IFNs, BLyS, IL-12p40, and stem cell factor/c-Kit ligand. Further, severely decreased levels of IL-1Ra in SLE patients compared with ANA-positive individuals may contribute to disease development. These results highlight the importance of IFN-related pathways and regulatory elements in SLE pathogenesis.

  View details for DOI 10.1002/art.39706

  View details for PubMedID 27059145

  View details for PubMedCentralID PMC5042816

• African American and European American SLE Patients with Variable Disease Activity Reveal Distinct Differences in CD4+T Cell and Monocyte Pathways Slight-Webb, S., Lu, R., Bean, K. M., Maecker, H. T., Utz, P. J., Guthridge, J. M., James, J. A. WILEY. 2016

  View details for Web of Science ID 000417143403003

• Whole Blood Phenotyping and Innate and Adaptive Stimulation Reveal Unique Differences in Granulocytes and Innate Pathways of African American SLE Patients with Variable Disease Activity Slight-Webb, S., Bean, K. M., Kheir, J., Adebayo, B., Maecker, H. T., Utz, P. J., James, J. A., Guthridge, J. M. WILEY. 2016

  View details for Web of Science ID 000417143404491

• Nucleic Acid-Targeting Pathways Promote Inflammation in Obesity-Related Insulin Resistance. Cell reports Revelo, X. S., Ghazarian, M., Chng, M. H., Luck, H., Kim, J. H., Zeng, K., Shi, S. Y., Tsai, S., Lei, H., Kenkel, J., Liu, C. L., Tangsombatvisit, S., Tsui, H., Sima, C., Xiao, C., Shen, L., Li, X., Jin, T., Lewis, G. F., Woo, M., Utz, P. J., Glogauer, M., Engleman, E., Winer, S., Winer, D. A. 2016; 16 (3): 717-730

  Abstract

  Obesity-related inflammation of metabolic tissues, including visceral adipose tissue (VAT) and liver, are key factors in the development of insulin resistance (IR), though many of the contributing mechanisms remain unclear. We show that nucleic-acid-targeting pathways downstream of extracellular trap (ET) formation, unmethylated CpG DNA, or ribonucleic acids drive inflammation in IR. High-fat diet (HFD)-fed mice show increased release of ETs in VAT, decreased systemic clearance of ETs, and increased autoantibodies against conserved nuclear antigens. In HFD-fed mice, this excess of nucleic acids and related protein antigens worsens metabolic parameters through a number of mechanisms, including activation of VAT macrophages and expansion of plasmacytoid dendritic cells (pDCs) in the liver. Consistently, HFD-fed mice lacking critical responders of nucleic acid pathways, Toll-like receptors (TLR)7 and TLR9, show reduced metabolic inflammation and improved glucose homeostasis. Treatment of HFD-fed mice with inhibitors of ET formation or a TLR7/9 antagonist improves metabolic disease. These findings reveal a pathogenic role for nucleic acid targeting as a driver of metabolic inflammation in IR.

  View details for DOI 10.1016/j.celrep.2016.06.024

  View details for PubMedID 27373163

• Multiplex giant magnetoresistive biosensor microarrays identify interferon-associated autoantibodies in systemic lupus erythematosus SCIENTIFIC REPORTS Lee, J., Haddon, D. J., Wand, H. E., Price, J. V., Diep, V. K., Hall, D. A., Petri, M., Baechler, E. C., Balboni, I. M., Utz, P. J., Wang, S. X. 2016; 6

  Abstract

  High titer, class-switched autoantibodies are a hallmark of systemic lupus erythematosus (SLE). Dysregulation of the interferon (IFN) pathway is observed in individuals with active SLE, although the association of specific autoantibodies with chemokine score, a combined measurement of three IFN-regulated chemokines, is not known. To identify autoantibodies associated with chemokine score, we developed giant magnetoresistive (GMR) biosensor microarrays, which allow the parallel measurement of multiple serum antibodies to autoantigens and peptides. We used the microarrays to analyze serum samples from SLE patients and found individuals with high chemokine scores had significantly greater reactivity to 13 autoantigens than individuals with low chemokine scores. Our findings demonstrate that multiple autoantibodies, including antibodies to U1-70K and modified histone H2B tails, are associated with IFN dysregulation in SLE. Further, they show the microarrays are capable of identifying autoantibodies associated with relevant clinical manifestations of SLE, with potential for use as biomarkers in clinical practice.

  View details for DOI 10.1038/srep27623

  View details for Web of Science ID 000377358700002

  View details for PubMedID 27279139

• Development of Th17-Associated Interstitial Kidney Inflammation in Lupus-Prone Mice Lacking the Gene Encoding STAT-1 ARTHRITIS & RHEUMATOLOGY Yiu, G., Rasmussen, T. K., Ajami, B., Haddon, D. J., Chu, A. D., Tangsombatvisit, S., Haynes, W. A., Diep, V., Steinman, L., Faix, J., Utz, P. J. 2016; 68 (5): 1233-1244

  Abstract

  Type I interferon (IFN) signaling is a central pathogenic pathway in systemic lupus erythematosus (SLE), and therapeutics targeting type I IFN signaling are in development. Multiple proteins with overlapping functions play a role in IFN signaling, but the signaling events downstream of receptor engagement are unclear. This study was undertaken to investigate the roles of the type I and type II IFN signaling components IFN-α/β/ω receptor 2 (IFNAR-2), IFN regulatory factor 9 (IRF-9), and STAT-1 in a mouse model of SLE.We used immunohistochemical staining and highly multiplexed assays to characterize pathologic changes in histology, autoantibody production, cytokine/chemokine profiles, and STAT phosphorylation in order to investigate the individual roles of IFNAR-2, IRF-9, and STAT-1 in MRL/lpr mice.We found that STAT-1(-/-) mice, but not IRF-9(-/-) or IFNAR-2(-/-) mice, developed interstitial nephritis characterized by infiltration with retinoic acid receptor-related orphan nuclear receptor γt-positive lymphocytes, macrophages, and eosinophils. Despite pronounced interstitial kidney disease and abnormal kidney function, STAT-1(-/-) mice had decreased proteinuria, glomerulonephritis, and autoantibody production. Phosphospecific flow cytometry revealed shunting of STAT phosphorylation from STAT-1 to STAT-3/4.We describe unique contributions of STAT-1 to pathology in different kidney compartments in a mouse model, and provide potentially novel insight into tubulointerstitial nephritis, a poorly understood complication that predicts end-stage kidney disease in SLE patients.

  View details for DOI 10.1002/art.39535

  View details for Web of Science ID 000375551600023

  View details for PubMedID 26636548

  View details for PubMedCentralID PMC4848130

• Anti-Insulin Immune Responses Are Detectable in Dogs with Spontaneous Diabetes PLOS ONE Kim, J., Furrow, E., Ritt, M. G., Utz, P. J., Robinson, W. H., Yu, L., Eckert, A., Stuebner, K., O'Brien, T. D., Steinman, L., Modiano, J. F. 2016; 11 (3)

  Abstract

  Diabetes mellitus occurs spontaneously in dogs. Although canine diabetes shares many features with human type-1 diabetes, there are differences that have cast doubt on the immunologic origin of the canine disease. In this study, we examined whether peripheral immune responses directed against islet antigens were present in dogs with diabetes. Routine diagnostics were used to confirm diabetic status, and serum samples from dogs with (N = 15) and without (N = 15) diabetes were analyzed for the presence of antibodies against islet antigens (insulin, glutamic acid decarboxylase, insulinoma-associated protein tyrosine phosphatase, and islet beta-cell zinc cation efflux transporter) using standard radioassays. Interferon-γ production from peripheral blood T cells stimulated by porcine insulin and by human insulin was tested using Elispot assays. Anti-insulin antibodies were detectable in a subset of diabetic dogs receiving insulin therapy. Pre-activated T cells and incipient insulin-reactive T cells in response to porcine or human insulin were identified in non-diabetic dogs and in dogs with diabetes. The data show that humoral and cellular anti-insulin immune responses are detectable in dogs with diabetes. This in turn provides support for the potential to ethically use dogs with diabetes to study the therapeutic potential of antigen-specific tolerance.

  View details for DOI 10.1371/journal.pone.0152397

  View details for Web of Science ID 000373121800066

  View details for PubMedID 27031512

  View details for PubMedCentralID PMC4816536

• Identification of Candidate Tolerogenic CD8(+) T Cell Epitopes for Therapy of Type 1 Diabetes in the NOD Mouse Model. Journal of diabetes research Yu, C., Burns, J. C., Robinson, W. H., Utz, P. J., Ho, P. P., Steinman, L., Frey, A. B. 2016; 2016: 9083103-?

  Abstract

  Type 1 diabetes is an autoimmune disease in which insulin-producing pancreatic islet β cells are the target of self-reactive B and T cells. T cells reactive with epitopes derived from insulin and/or IGRP are critical for the initiation and maintenance of disease, but T cells reactive with other islet antigens likely have an essential role in disease progression. We sought to identify candidate CD8(+) T cell epitopes that are pathogenic in type 1 diabetes. Proteins that elicit autoantibodies in human type 1 diabetes were analyzed by predictive algorithms for candidate epitopes. Using several different tolerizing regimes using synthetic peptides, two new predicted tolerogenic CD8(+) T cell epitopes were identified in the murine homolog of the major human islet autoantigen zinc transporter ZnT8 (aa 158-166 and 282-290) and one in a non-β cell protein, dopamine β-hydroxylase (aa 233-241). Tolerizing vaccination of NOD mice with a cDNA plasmid expressing full-length proinsulin prevented diabetes, whereas plasmids encoding ZnT8 and DβH did not. However, tolerizing vaccination of NOD mice with the proinsulin plasmid in combination with plasmids expressing ZnT8 and DβH decreased insulitis and enhanced prevention of disease compared to vaccination with the plasmid encoding proinsulin alone.

  View details for DOI 10.1155/2016/9083103

  View details for PubMedID 27069933

  View details for PubMedCentralID PMC4812430

• Protein microarrays identify disease-specific anti-cytokine autoantibody profiles in the landscape of immunodeficiency JOURNAL OF ALLERGY AND CLINICAL IMMUNOLOGY Rosenberg, J. M., Price, J. V., Barcenas-Morales, G., Ceron-Gutierrez, L., Davies, S., Kumararatne, D. S., Doeffinger, R., Utz, P. J. 2016; 137 (1): 204-?

  Abstract

  Anti-cytokine autoantibodies (ACAAs) are pathogenic in a handful of rare immunodeficiencies. However, the prevalence and significance of other ACAAs across immunodeficiencies have not yet been described.We profiled ACAAs in a diverse cohort of serum samples from patients with immunodeficiency and assessed the sensitivity and specificity of protein microarrays for ACAA identification and discovery.Highly multiplexed protein microarrays were designed and fabricated. Blinded serum samples from a cohort of 58 immunodeficiency patients and healthy control subjects were used to probe microarrays. Unsupervised hierarchical clustering was used to identify clusters of reactivity, and after unblinding, significance analysis of microarrays was used to identify disease-specific autoantibodies. A bead-based assay was used to validate protein microarray results. Blocking activity of serum containing ACAAs was measured in vitro.Protein microarrays were highly sensitive and specific for the detection of ACAAs in patients with autoimmune polyendocrine syndrome type I and pulmonary alveolar proteinosis, detecting ACAA levels consistent with those reported in the published literature. Protein microarray results were validated by using an independent bead-based assay. To confirm the functional significance of these ACAAs, we tested and confirmed the blocking activity of select ACAAs in vitro.Protein microarrays are a powerful tool for ACAA detection and discovery, and they hold promise as a diagnostic for the evaluation and monitoring of clinical immunodeficiency.

  View details for DOI 10.1016/j.jaci.2015.07.032

  View details for Web of Science ID 000367724300012

• Protein Microarrays Identify Disease Specific Anti-Cytokine Autoantibody Profiles In The Landscape Of Immunodeficiency Rosenberg, J., Price, J., Barcenas-Morales, G., Ceron-Gutierrez, L., Davies, S., Kumararatne, D., Doffinger, R., Utz, P. WILEY-BLACKWELL. 2015: 3

  View details for Web of Science ID 000367267800007

• Mapping epitopes of U1-70K autoantibodies at single-amino acid resolution AUTOIMMUNITY Haddon, D. J., Jarrell, J. A., Diep, V. K., Wand, H. E., Price, J. V., Tangsombatvisit, S., Credo, G. M., Mackey, S., Dekker, C. L., Baechler, E. C., Liu, C. L., Varma, M., Utz, P. J. 2015; 48 (8): 513-523

  Abstract

  The mechanisms underlying development of ribonucleoprotein (RNP) autoantibodies are unclear. The U1-70K protein is the predominant target of RNP autoantibodies, and the RNA binding domain has been shown to be the immunodominant autoantigenic region of U1-70K, although the specific epitopes are not known. To precisely map U1-70K epitopes, we developed silicon-based peptide microarrays with >5700 features, corresponding to 843 unique peptides derived from the U1-70K protein. The microarrays feature overlapping peptides, with single-amino acid resolution in length and location, spanning amino acids 110-170 within the U1-70K RNA binding domain. We evaluated the serum IgG of a cohort of patients with systemic lupus erythematosus (SLE; n = 26) using the microarrays, and identified multiple reactive epitopes, including peptides 116-121 and 143-148. Indirect peptide ELISA analysis of the sera of patients with SLE (n = 88) revealed that ∼14% of patients had serum IgG reactivity to 116-121, while reactivity to 143-148 appeared to be limited to a single patient. SLE patients with serum reactivity to 116-121 had significantly lower SLE Disease Activity Index (SLEDAI) scores at the time of sampling, compared to non-reactive patients. Minimal reactivity to the peptides was observed in the sera of healthy controls (n = 92). Competitive ELISA showed antibodies to 116-121 bind a common epitope in U1-70K (68-72) and the matrix protein M1 of human influenza B viruses. Institutional Review Boards approved this study. Knowledge of the precise epitopes of U1-70K autoantibodies may provide insight into the mechanisms of development of anti-RNP, identify potential clinical biomarkers and inform ongoing clinical trails of peptide-based therapeutics.

  View details for DOI 10.3109/08916934.2015.1077233

  View details for Web of Science ID 000366755000003

  View details for PubMedID 26333287

• Single-cell systems-level analysis of human Toll-like receptor activation defines a chemokine signature in patients with systemic lupus erythematosus JOURNAL OF ALLERGY AND CLINICAL IMMUNOLOGY O'Gorman, W. E., Hsieh, E. W., Savig, E. S., Gherardini, P. F., Hernandez, J. D., Hansmann, L., Balboni, I. M., Utz, P. J., Bendall, S. C., Fantl, W. J., Lewis, D. B., Nolan, G. P., Davis, M. M. 2015; 136 (5): 1326-1336

  Abstract

  Activation of Toll-like receptors (TLRs) induces inflammatory responses involved in immunity to pathogens and autoimmune pathogenesis, such as in patients with systemic lupus erythematosus (SLE). Although TLRs are differentially expressed across the immune system, a comprehensive analysis of how multiple immune cell subsets respond in a system-wide manner has not been described.We sought to characterize TLR activation across multiple immune cell subsets and subjects, with the goal of establishing a reference framework against which to compare pathologic processes.Peripheral whole-blood samples were stimulated with TLR ligands and analyzed by means of mass cytometry simultaneously for surface marker expression, activation states of intracellular signaling proteins, and cytokine production. We developed a novel data visualization tool to provide an integrated view of TLR signaling networks with single-cell resolution. We studied 17 healthy volunteer donors and 8 patients with newly diagnosed and untreated SLE.Our data revealed the diversity of TLR-induced responses within cell types, with TLR ligand specificity. Subsets of natural killer cells and T cells selectively induced nuclear factor κ light chain enhancer of activated B cells in response to TLR2 ligands. CD14(hi) monocytes exhibited the most polyfunctional cytokine expression patterns, with more than 80 distinct cytokine combinations. Monocytic TLR-induced cytokine patterns were shared among a group of healthy donors, with minimal intraindividual and interindividual variability. Furthermore, autoimmune disease altered baseline cytokine production; newly diagnosed untreated SLE patients shared a distinct monocytic chemokine signature, despite clinical heterogeneity.Mass cytometry defined a systems-level reference framework for human TLR activation, which can be applied to study perturbations in patients with inflammatory diseases, such as SLE.

  View details for DOI 10.1016/j.jaci.2015.04.008

  View details for Web of Science ID 000364787200023

  View details for PubMedID 26037552

• Broad-spectrum antibodies against self-antigens and cytokines in RAG deficiency JOURNAL OF CLINICAL INVESTIGATION Walter, J. E., Rosen, L. B., Csomos, K., Rosenberg, J. M., Mathew, D., Keszei, M., Ujhazi, B., Chen, K., Lee, Y. N., Tirosh, I., Dobbs, K., Al-Herz, W., Cowan, M. J., Puck, J., Bleesing, J. J., Grimley, M. S., Malech, H., De Ravin, S. S., Gennery, A. R., Abraham, R. S., Joshi, A. Y., Boyce, T. G., Butte, M. J., Nadeau, K. C., Balboni, I., Sullivan, K. E., Akhter, J., Adeli, M., El-Feky, R. A., El-Ghoneimy, D. H., Dbaibo, G., Wakim, R., Azzari, C., Palma, P., Cancrini, C., Capuder, K., Condino-Neto, A., Costa-Carvalho, B. T., Oliveira, J. B., Roifman, C., Buchbinder, D., Kumanovics, A., Luis Franco, J., Niehues, T., Schuetz, C., Kuijpers, T., Yee, C., Chou, J., Masaad, M. J., Geha, R., Uze, G., Gelman, R., Holland, S. M., Recher, M., Utz, P. J., Browne, S. K., Notarangelo, L. D. 2015; 125 (11): 4135-4148

  Abstract

  Patients with mutations of the recombination-activating genes (RAG) present with diverse clinical phenotypes, including severe combined immune deficiency (SCID), autoimmunity, and inflammation. However, the incidence and extent of immune dysregulation in RAG-dependent immunodeficiency have not been studied in detail. Here, we have demonstrated that patients with hypomorphic RAG mutations, especially those with delayed-onset combined immune deficiency and granulomatous/autoimmune manifestations (CID-G/AI), produce a broad spectrum of autoantibodies. Neutralizing anti-IFN-α or anti-IFN-ω antibodies were present at detectable levels in patients with CID-G/AI who had a history of severe viral infections. As this autoantibody profile is not observed in a wide range of other primary immunodeficiencies, we hypothesized that recurrent or chronic viral infections may precipitate or aggravate immune dysregulation in RAG-deficient hosts. We repeatedly challenged Rag1S723C/S723C mice, which serve as a model of leaky SCID, with agonists of the virus-recognizing receptors TLR3/MDA5, TLR7/-8, and TLR9 and found that this treatment elicits autoantibody production. Altogether, our data demonstrate that immune dysregulation is an integral aspect of RAG-associated immunodeficiency and indicate that environmental triggers may modulate the phenotypic expression of autoimmune manifestations.

  View details for DOI 10.1172/JCI80477

  View details for Web of Science ID 000364110000017

  View details for PubMedID 26457731

• Applications of Protein Microarray for Saliva Diagnostics in Autoimmune Diseases Lee, Y., Kim, Y., Hong, S., Utz, P. J. WILEY-BLACKWELL. 2015

  View details for Web of Science ID 000370860202526

• Divergent Phenotypic Patterns Between Systemic Lupus Erythematosus and Healthy Anti-Nuclear Antibody Positive Individuals Reveal Distinct Differences in B Cell and Myeloid Populations Among Ethnicities Slight-Webb, S., Lu, R., Maecker, H. T., Utz, P. J., Guthridge, J. M., James, J. A. WILEY-BLACKWELL. 2015

  View details for Web of Science ID 000370860202063

• Autoantigen microarrays reveal autoantibodies associated with proliferative nephritis and active disease in pediatric systemic lupus erythematosus ARTHRITIS RESEARCH & THERAPY Haddon, D. J., Diep, V. K., Price, J. V., Limb, C., Utz, P. J., Balboni, I. 2015; 17

  Abstract

  Pediatric systemic lupus erythematosus (pSLE) patients often initially present with more active and severe disease than adults, including a higher frequency of lupus nephritis. Specific autoantibodies, including anti-C1q, anti-DNA and anti-alpha-actinin, have been associated with kidney involvement in SLE, and DNA antibodies are capable of initiating early-stage lupus nephritis in severe combined immunodeficiency (SCID) mice. Over 100 different autoantibodies have been described in SLE patients, highlighting the need for comprehensive autoantibody profiling. Knowledge of the antibodies associated with pSLE and proliferative nephritis will increase the understanding of SLE pathogenesis, and may aid in monitoring patients for renal flare.We used autoantigen microarrays composed of 140 recombinant or purified antigens to compare the serum autoantibody profiles of new-onset pSLE patients (n = 45) to healthy controls (n = 17). We also compared pSLE patients with biopsy-confirmed class III or IV proliferative nephritis (n = 23) and without significant renal involvement (n = 18). We performed ELISA with selected autoantigens to validate the microarray findings. We created a multiple logistic regression model, based on the ELISA and clinical information, to predict whether a patient had proliferative nephritis, and used a validation cohort (n = 23) and longitudinal samples (88 patient visits) to test its accuracy.Fifty autoantibodies were at significantly higher levels in the sera of pSLE patients compared to healthy controls, including anti-B cell-activating factor (BAFF). High levels of anti-BAFF were associated with active disease. Thirteen serum autoantibodies were present at significantly higher levels in pSLE patients with proliferative nephritis than those without, and we confirmed five autoantigens (dsDNA, C1q, collagens IV and X and aggrecan) by ELISA. Our model, based on ELISA measurements and clinical variables, correctly identified patients with proliferative nephritis with 91 % accuracy.Autoantigen microarrays are an ideal platform for identifying autoantibodies associated with both pSLE and specific clinical manifestations of pSLE. Using multiple regression analysis to integrate autoantibody and clinical data permits accurate prediction of clinical manifestations with complex etiologies in pSLE.

  View details for DOI 10.1186/s13075-015-0682-6

  View details for Web of Science ID 000357456500001

  View details for PubMedID 26081107

  View details for PubMedCentralID PMC4493823

• Gene expression changes reflect clinical response in a placebo-controlled randomized trial of abatacept in patients with diffuse cutaneous systemic sclerosis ARTHRITIS RESEARCH & THERAPY Chakravarty, E. F., Martyanov, V., Fiorentino, D., Wood, T. A., Haddon, D. J., Jarrell, J. A., Utz, P. J., Genovese, M. C., Whitfield, M. L., Chung, L. 2015; 17

  Abstract

  Systemic sclerosis is an autoimmune disease characterized by inflammation and fibrosis of the skin and internal organs. We sought to assess the clinical and molecular effects associated with response to intravenous abatacept in patients with diffuse cutaneous systemic.Adult diffuse cutaneous systemic sclerosis patients were randomized in a 2:1 double-blinded fashion to receive abatacept or placebo over 24 weeks. Primary outcomes were safety and the change in modified Rodnan Skin Score (mRSS) at week 24 compared with baseline. Improvers were defined as patients with a decrease in mRSS of ≥30% post-treatment compared to baseline. Skin biopsies were obtained for differential gene expression and pathway enrichment analyses and intrinsic gene expression subset assignment.Ten subjects were randomized to abatacept (n = 7) or placebo (n = 3). Disease duration from first non-Raynaud's symptom was significantly longer (8.8 ± 3.8 years vs. 2.4 ± 1.6 years, p = 0.004) and median mRSS was higher (30 vs. 22, p = 0.05) in the placebo compared to abatacept group. Adverse events were similar in the two groups. Five out of seven patients (71%) randomized to abatacept and one out of three patients (33%) randomized to placebo experienced ≥30% improvement in skin score. Subjects receiving abatacept showed a trend toward improvement in mRSS at week 24 (-8.6 ± 7.5, p = 0.0625) while those in the placebo group did not (-2.3 ± 15, p = 0.75). After adjusting for disease duration, mRSS significantly improved in the abatacept compared with the placebo group (abatacept vs. placebo mRSS decrease estimate -9.8, 95% confidence interval -16.7 to -3.0, p = 0.0114). In the abatacept group, the patients in the inflammatory intrinsic subset showed a trend toward greater improvement in skin score at 24 weeks compared with the patients in the normal-like intrinsic subset (-13.5 ± 3.1 vs. -4.5 ± 6.4, p = 0.067). Abatacept resulted in decreased CD28 co-stimulatory gene expression in improvers consistent with its mechanism of action. Improvers mapped to the inflammatory intrinsic subset and showed decreased gene expression in inflammatory pathways, while non-improver and placebos showed stable or reverse gene expression over 24 weeks.Clinical improvement following abatacept therapy was associated with modulation of inflammatory pathways in skin.ClinicalTrials.gov NCT00442611. Registered 1 March 2007.

  View details for DOI 10.1186/s13075-015-0669-3

  View details for Web of Science ID 000357069900001

  View details for PubMedID 26071192

  View details for PubMedCentralID PMC4487200

• Comparison of systemic lupus erythematosus and healthy anti-nuclear antibody positive African-Americans reveals distinct differences in CD4+and CD8+T cell populations Lu, R., Slight-Webb, S., Maecker, H., Utz, P., Guthridge, J., James, J. AMER ASSOC IMMUNOLOGISTS. 2015

  View details for Web of Science ID 000379404500238

• Regulation of ribosomal RNA synthesis in T cells: requirement for GTP and Ebp1. Blood Nguyen, L. X., Lee, Y., Urbani, L., Utz, P. J., Hamburger, A. W., Sunwoo, J. B., Mitchell, B. S. 2015; 125 (16): 2519-2529

  Abstract

  Mycophenolic acid (MPA) is the active metabolite of Mycophenolate Mofeteil (MMF), an effective immunosuppressive drug. Both MPA and MMF are highly specific inhibitors of guanine nucleotide synthesis and of T cell activation. However, the mechanism by which guanine nucleotide depletion suppresses T cell activation is unknown. Depletion of GTP inhibits ribosomal RNA synthesis in T cells by inhibiting TIF-IA, a GTP-binding protein that recruits RNA Polymerase I to the ribosomal DNA promoter. TIF-IA-GTP binds the ErbB3 binding protein 1 (Ebp1) and together they enhance the transcription of proliferating cell nuclear antigen (PCNA). GTP binding by TIF-IA and Ebp1 phosphorylation by protein kinase C delta are both required for optimal PCNA expression. The PKC inhibitor Sotrastaurin markedly potentiates the inhibition of rRNA synthesis, PCNA expression, and T cell activation induced by MPA, suggesting that the combination of the two agents are more highly effective than either alone in inducing immunosuppression.

  View details for DOI 10.1182/blood-2014-12-616433

  View details for PubMedID 25691158

• Regulation of ribosomal RNA synthesis in T cells: requirement for GTP and Ebp1 BLOOD Le Xuan Truong Nguyen, L. X., Lee, Y., Urbani, L., Utz, P. J., Hamburger, A. W., Sunwoo, J. B., Mitchell, B. S. 2015; 125 (16): 2519-2529

  View details for DOI 10.1182/blood-2014-12-616433

  View details for Web of Science ID 000354751700013

• Protein microarrays: a new tool for the study of autoantibodies in immunodeficiency FRONTIERS IN IMMUNOLOGY Rosenberg, J. M., Utz, P. J. 2015; 6: 1-8

  Abstract

  Autoimmunity is highly coincident with immunodeficiency. In a small but growing number of primary immunodeficiencies, autoantibodies are diagnostic of a given disease and implicated in disease pathogenesis. In order to improve our understanding of the role of autoantibodies in immunodeficiencies and to discover novel autoantibodies, new proteomic tools are needed. Protein microarrays have the ability to screen for reactivity to hundreds to many thousands of unique autoantigens simultaneously on a single chip using minimal serum input. Here, we review different types of protein microarrays and how they can be useful in framing the study of primary and secondary immunodeficiencies.

  View details for DOI 10.3389/fimmu.2015.00138

  View details for Web of Science ID 000354923200001

• COMMON VARIABLE IMMUNODEFICIENCY AND EARLY-ONSET AUTOIMMUNE MANIFESTATIONS ASSOCIATED WITH COUPLED MUTATIONS IN NFKB2 AND STAT5A GENES Maccari, M., Floris, M., Corrente, S., Angius, A., Sartirana, C., Di Cesare, S., Rosenberg, J. M., Utz, P. J., Bulfone, A., Angelini, F., Fierabracci, A., Cambiaso, P., Aiuti, A., Cappa, M., Cancrini, C., Bacchetta, R. SPRINGER/PLENUM PUBLISHERS. 2015: 317

  View details for Web of Science ID 000351510200052

• 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 Kattah, N. H., Newell, E. W., Jarrell, J. A., Chu, A. D., Xie, J., Kattah, M. G., Goldberger, O., Ye, J., Chakravarty, E. F., Davis, M. M., Utz, P. J. 2015; 112 (10): 3044-3049

  Abstract

  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

  View details for PubMedCentralID PMC4364210

• Autoantigen microarrays reveal autoantibodies associated with proliferative nephritis and active disease in pediatric systemic lupus erythematosus. Arthritis research & therapy Haddon, D. J., Diep, V. K., Price, J. V., Limb, C., Utz, P. J., Balboni, I. 2015; 17: 162-?

  Abstract

  Pediatric systemic lupus erythematosus (pSLE) patients often initially present with more active and severe disease than adults, including a higher frequency of lupus nephritis. Specific autoantibodies, including anti-C1q, anti-DNA and anti-alpha-actinin, have been associated with kidney involvement in SLE, and DNA antibodies are capable of initiating early-stage lupus nephritis in severe combined immunodeficiency (SCID) mice. Over 100 different autoantibodies have been described in SLE patients, highlighting the need for comprehensive autoantibody profiling. Knowledge of the antibodies associated with pSLE and proliferative nephritis will increase the understanding of SLE pathogenesis, and may aid in monitoring patients for renal flare.We used autoantigen microarrays composed of 140 recombinant or purified antigens to compare the serum autoantibody profiles of new-onset pSLE patients (n = 45) to healthy controls (n = 17). We also compared pSLE patients with biopsy-confirmed class III or IV proliferative nephritis (n = 23) and without significant renal involvement (n = 18). We performed ELISA with selected autoantigens to validate the microarray findings. We created a multiple logistic regression model, based on the ELISA and clinical information, to predict whether a patient had proliferative nephritis, and used a validation cohort (n = 23) and longitudinal samples (88 patient visits) to test its accuracy.Fifty autoantibodies were at significantly higher levels in the sera of pSLE patients compared to healthy controls, including anti-B cell-activating factor (BAFF). High levels of anti-BAFF were associated with active disease. Thirteen serum autoantibodies were present at significantly higher levels in pSLE patients with proliferative nephritis than those without, and we confirmed five autoantigens (dsDNA, C1q, collagens IV and X and aggrecan) by ELISA. Our model, based on ELISA measurements and clinical variables, correctly identified patients with proliferative nephritis with 91 % accuracy.Autoantigen microarrays are an ideal platform for identifying autoantibodies associated with both pSLE and specific clinical manifestations of pSLE. Using multiple regression analysis to integrate autoantibody and clinical data permits accurate prediction of clinical manifestations with complex etiologies in pSLE.

  View details for DOI 10.1186/s13075-015-0682-6

  View details for PubMedID 26081107

• Protein microarrays: a new tool for the study of autoantibodies in immunodeficiency. Frontiers in immunology Rosenberg, J. M., Utz, P. J. 2015; 6: 138-?

  Abstract

  Autoimmunity is highly coincident with immunodeficiency. In a small but growing number of primary immunodeficiencies, autoantibodies are diagnostic of a given disease and implicated in disease pathogenesis. In order to improve our understanding of the role of autoantibodies in immunodeficiencies and to discover novel autoantibodies, new proteomic tools are needed. Protein microarrays have the ability to screen for reactivity to hundreds to many thousands of unique autoantigens simultaneously on a single chip using minimal serum input. Here, we review different types of protein microarrays and how they can be useful in framing the study of primary and secondary immunodeficiencies.

  View details for DOI 10.3389/fimmu.2015.00138

  View details for PubMedID 25904912

• Overexpression of microRNA-155 increases IL-21 mediated STAT3 signaling and IL-21 production in systemic lupus erythematosus. Arthritis research & therapy Rasmussen, T. K., Andersen, T., Bak, R. O., Yiu, G., Sørensen, C. M., Stengaard-Pedersen, K., Mikkelsen, J. G., Utz, P. J., Holm, C. K., Deleuran, B. 2015; 17: 154-?

  Abstract

  Interleukin (IL-)21 is a key cytokine in autoimmune diseases such as systemic lupus erythematosus (SLE) by its regulation of autoantibody production and inflammatory responses. The objective of this study is to investigate the signaling capacity of IL-21 in T and B cells and assess its possible regulation by microRNA (miR)-155 and its target gene suppressor of cytokine signaling 1 (SOCS1) in SLE.The signaling capacity of IL-21 was quantified by stimulating peripheral blood monocuclear cells (PBMCs) with IL-21 and measuring phosphorylation of STAT3 (pSTAT3) in CD4+ T cells, B cells, and NK cells. Induction of miR-155 by IL-21 was investigated by stimulating purified CD4+ T cells with IL-21 and measuring miR-155 expression levels. The functional role of miR-155 was assessed by overexpressing miR-155 in PBMCs from SLE patients and healthy controls (HCs) and measuring its effects on STAT3 and IL-21 production in CD4+ and CD8+ T cells.Induction of pSTAT3 in CD4+ T cells in response to IL-21 was significantly decreased in SLE patients compared to HCs (p < 0.0001). Further, expression levels of miR-155 were significantly decreased and SOCS1 correspondingly increased in CD4+ T cells from SLE patients. Finally, overexpression of miR-155 in CD4+ T cells increased STAT3 phosphorylation in response to IL-21 treatment (p < 0.01) and differentially increased IL-21 production in SLE patients compared to HCs (p < 0.01).We demonstrate that SLE patients have reduced IL-21 signaling capacity, decreased miR-155 levels, and increased SOCS1 levels compared to HCs. The reduced IL-21 signaling in SLE could be rescued by overexpression of miR-155, suggesting an important role for miR-155 in the reduced IL-21 signaling observed in SLE.

  View details for DOI 10.1186/s13075-015-0660-z

  View details for PubMedID 26055806

• Overexpression of microRNA-155 increases IL-21 mediated STAT3 signaling and IL-21 production in systemic lupus erythematosus. Arthritis research & therapy Rasmussen, T. K., Andersen, T., Bak, R. O., Yiu, G., Sørensen, C. M., Stengaard-Pedersen, K., Mikkelsen, J. G., Utz, P. J., Holm, C. K., Deleuran, B. 2015; 17: 154-?

  Abstract

  Interleukin (IL-)21 is a key cytokine in autoimmune diseases such as systemic lupus erythematosus (SLE) by its regulation of autoantibody production and inflammatory responses. The objective of this study is to investigate the signaling capacity of IL-21 in T and B cells and assess its possible regulation by microRNA (miR)-155 and its target gene suppressor of cytokine signaling 1 (SOCS1) in SLE.The signaling capacity of IL-21 was quantified by stimulating peripheral blood monocuclear cells (PBMCs) with IL-21 and measuring phosphorylation of STAT3 (pSTAT3) in CD4+ T cells, B cells, and NK cells. Induction of miR-155 by IL-21 was investigated by stimulating purified CD4+ T cells with IL-21 and measuring miR-155 expression levels. The functional role of miR-155 was assessed by overexpressing miR-155 in PBMCs from SLE patients and healthy controls (HCs) and measuring its effects on STAT3 and IL-21 production in CD4+ and CD8+ T cells.Induction of pSTAT3 in CD4+ T cells in response to IL-21 was significantly decreased in SLE patients compared to HCs (p < 0.0001). Further, expression levels of miR-155 were significantly decreased and SOCS1 correspondingly increased in CD4+ T cells from SLE patients. Finally, overexpression of miR-155 in CD4+ T cells increased STAT3 phosphorylation in response to IL-21 treatment (p < 0.01) and differentially increased IL-21 production in SLE patients compared to HCs (p < 0.01).We demonstrate that SLE patients have reduced IL-21 signaling capacity, decreased miR-155 levels, and increased SOCS1 levels compared to HCs. The reduced IL-21 signaling in SLE could be rescued by overexpression of miR-155, suggesting an important role for miR-155 in the reduced IL-21 signaling observed in SLE.

  View details for DOI 10.1186/s13075-015-0660-z

  View details for PubMedID 26055806

• Measurement of mast cell surface molecules by high-throughput immunophenotyping using transcription (HIT). Methods in molecular biology (Clifton, N.J.) Haddon, D. J., Jarrell, J. A., Hughes, M. R., Snyder, K., McNagny, K. M., Kattah, M. G., Utz, P. J. 2015; 1220: 381-400

  Abstract

  Here we describe the application of a highly multiplexed proteomic assay, called HIT (high-throughput immunophenotyping using transcription), to analyze human mast cell surface antigens at rest and during stimulation. HIT allows analysis of up to 100 analytes, including surface antigens and intracellular phosphoproteins, transcription factors, and cytokines, in a single experiment. Briefly, anti-mouse monovalent Fab fragments are covalently conjugated with barcoded oligonucleotides to generate a panel of conjugates. The oligonucleotide-Fab fragment conjugates are bound to monoclonal primary antibodies, creating a cocktail of up to 48 unique barcoded primary antibodies. As few as 100,000 mast cells are stained with the cocktail and the barcodes of the bound primary antibodies are amplified by in vitro transcription with fluorescently labeled NTPs. The resulting barcoded transcripts are quantified using a microarray spotted with oligonucleotides that are complementary to the barcoded transcripts. Differences in levels of the barcoded transcripts correlate well with actual protein levels and are capable of detecting stimulation-dependent changes in protein levels. HIT is an invaluable, broad-spectrum approach for characterizing mast cell surface antigens, signaling molecules, transcription factors, and cytokines.

  View details for DOI 10.1007/978-1-4939-1568-2_24

  View details for PubMedID 25388264

• Gene expression changes reflect clinical response in a placebo-controlled randomized trial of abatacept in patients with diffuse cutaneous systemic sclerosis. Arthritis research & therapy Chakravarty, E. F., Martyanov, V., Fiorentino, D., Wood, T. A., Haddon, D. J., Jarrell, J. A., Utz, P. J., Genovese, M. C., Whitfield, M. L., Chung, L. 2015; 17: 159-?

  Abstract

  Systemic sclerosis is an autoimmune disease characterized by inflammation and fibrosis of the skin and internal organs. We sought to assess the clinical and molecular effects associated with response to intravenous abatacept in patients with diffuse cutaneous systemic.Adult diffuse cutaneous systemic sclerosis patients were randomized in a 2:1 double-blinded fashion to receive abatacept or placebo over 24 weeks. Primary outcomes were safety and the change in modified Rodnan Skin Score (mRSS) at week 24 compared with baseline. Improvers were defined as patients with a decrease in mRSS of ≥30% post-treatment compared to baseline. Skin biopsies were obtained for differential gene expression and pathway enrichment analyses and intrinsic gene expression subset assignment.Ten subjects were randomized to abatacept (n = 7) or placebo (n = 3). Disease duration from first non-Raynaud's symptom was significantly longer (8.8 ± 3.8 years vs. 2.4 ± 1.6 years, p = 0.004) and median mRSS was higher (30 vs. 22, p = 0.05) in the placebo compared to abatacept group. Adverse events were similar in the two groups. Five out of seven patients (71%) randomized to abatacept and one out of three patients (33%) randomized to placebo experienced ≥30% improvement in skin score. Subjects receiving abatacept showed a trend toward improvement in mRSS at week 24 (-8.6 ± 7.5, p = 0.0625) while those in the placebo group did not (-2.3 ± 15, p = 0.75). After adjusting for disease duration, mRSS significantly improved in the abatacept compared with the placebo group (abatacept vs. placebo mRSS decrease estimate -9.8, 95% confidence interval -16.7 to -3.0, p = 0.0114). In the abatacept group, the patients in the inflammatory intrinsic subset showed a trend toward greater improvement in skin score at 24 weeks compared with the patients in the normal-like intrinsic subset (-13.5 ± 3.1 vs. -4.5 ± 6.4, p = 0.067). Abatacept resulted in decreased CD28 co-stimulatory gene expression in improvers consistent with its mechanism of action. Improvers mapped to the inflammatory intrinsic subset and showed decreased gene expression in inflammatory pathways, while non-improver and placebos showed stable or reverse gene expression over 24 weeks.Clinical improvement following abatacept therapy was associated with modulation of inflammatory pathways in skin.ClinicalTrials.gov NCT00442611. Registered 1 March 2007.

  View details for DOI 10.1186/s13075-015-0669-3

  View details for PubMedID 26071192

• Vimentin Is a Dominant Target of In Situ Humoral Immunity in Human Lupus Tubulointerstitial Nephritis ARTHRITIS & RHEUMATOLOGY Kinloch, A. J., Chang, A., Ko, K., dunand, C. J., Henderson, S., Maienschein-Cline, M., Kaverina, N., Rovin, B. H., Ferrer, M. S., Wolfgeher, D., Liarski, V., Haddon, D. J., Utz, P. J., Wilson, P. C., Clark, M. R. 2014; 66 (12): 3359-3370

  Abstract

  In lupus nephritis (LN), severe tubulointerstitial inflammation (TII) predicts progression to renal failure. Severe TII is associated with tertiary lymphoid neogenesis and in situ antigen-driven clonal B cell selection. The autoantigen(s) driving in situ B cell selection in TII are not known. This study was undertaken to identify the dominant driving autoantigen(s).Single CD38+ or Ki-67+ B cells were laser captured from 7 biopsy specimens that were diagnostic for LN. Eighteen clonally expanded immunoglobulin heavy- and light-chain variable region pairs were cloned and expressed as monoclonal antibodies. Seven more antibodies were cloned from flow-sorted CD38+ cells from an eighth biopsy specimen. Antigen characterization was performed using a combination of confocal microscopy, enzyme-linked immunosorbent assay, screening protoarrays, immunoprecipitation, and mass spectrometry. Serum IgG titers to the dominant antigen in 48 LN and 35 non-nephritic lupus samples were determined using purified antigen-coated arrays. Autoantigen expression on normal and LN kidney was localized by immunohistochemistry and immunofluorescence.Eleven of 25 antibodies reacted with cytoplasmic structures, 4 reacted with nuclei, and none reacted with double-stranded DNA. Vimentin was the only autoantigen identified by both mass spectrometry and protoarray. Ten of the 11 anticytoplasmic TII antibodies directly bound vimentin. Vimentin was highly expressed by tubulointerstitial inflammatory cells, and the TII antibodies tested preferentially bound inflamed tubulointerstitium. Finally, high titers of serum antivimentin antibodies were associated with severe TII (P = 0.0001).Vimentin, an antigenic feature of inflammation, is a dominant autoantigen targeted in situ in LN TII. This adaptive autoimmune response likely feeds forward to worsen TII and renal damage.

  View details for DOI 10.1002/art.38888

  View details for Web of Science ID 000345571200013

  View details for PubMedID 25306868

  View details for PubMedCentralID PMC4264660

• Luminex and Autoantigen Microarray Analysis of Sera from Patients with Diffuse Cutaneous Systemic Sclerosis Reveals Changes Associated with Imatinib Mesylate Treatment. Haddon, D., Wand, H., Utz, P. J., Spiera, R. F., Gordon, J. K., Chung, L. WILEY-BLACKWELL. 2014: S388

  View details for Web of Science ID 000344384901422

• Microrna-155 Suppresses IL-21 Signaling and Production in Systemic Lupus Erythematosus. Rasmussen, T. K., Andersen, T., Bak, R., Yiu, G., Steengaard-Petersen, K., Mikkelsen, J. G., Utz, P. J., Holm, C., Deleuran, B. WILEY-BLACKWELL. 2014: S1197

  View details for Web of Science ID 000344384905414

• Comparison of Systemic Lupus Erythematosus and Healthy Anti-Nuclear Antibody Positive African-Americans Reveals Distinct Differences in T Cell and Progenitor Populations Lu, R., Slight-Webb, S., Maecker, H. T., Utz, P. J., Guthridge, J. M., James, J. A. WILEY-BLACKWELL. 2014: S1176

  View details for Web of Science ID 000344384905365

• 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 Tang, X., Zhang, B., Jarrell, J. A., Price, J. V., Dai, H., Utz, P. J., Strober, S. 2014; 50: 87-98

  Abstract

  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 PubMedID 24508410

  View details for PubMedCentralID PMC4002579

• Broad-Scale Phosphoprotein Profiling of Beta Adrenergic Receptor (beta-AR) Signaling Reveals Novel Phosphorylation and Dephosphorylation Events PLOS ONE Chruscinski, A. J., Singh, H., Chan, S. M., Utz, P. J. 2013; 8 (12)

  Abstract

  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

  View details for PubMedCentralID PMC3855414

• Clinical optimization of antigen specific modulation of type 1 diabetes with the plasmid DNA platform CLINICAL IMMUNOLOGY Gottlieb, P., Utz, P. J., Robinson, W., Steinman, L. 2013; 149 (3): 297-306

  Abstract

  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

• Protein microarray analysis reveals BAFF-binding autoantibodies in systemic lupus erythematosus JOURNAL OF CLINICAL INVESTIGATION Price, J. V., Haddon, D. J., Kemmer, D., Delepine, G., Mandelbaum, G., Jarrell, J. A., Gupta, R., Balboni, I., Chakravarty, E. F., Sokolove, J., Shum, A. K., Anderson, M. S., Cheng, M. H., Robinson, W. H., Browne, S. K., Holland, S. M., Baechler, E. C., Utz, P. J. 2013; 123 (12): 5135-5145

  Abstract

  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

  View details for PubMedCentralID PMC3859403

• Autoantigen Microarray Analysis Of Sera From New-Onset Pediatric Systemic Lupus Erythematosus Patients: A Distinct Autoantibody Profile Associated With Class III/IV Lupus Nephritis Balboni, I., Haddon, D., Diep, V., Limb, C., Utz, P. WILEY-BLACKWELL. 2013: S1227

  View details for Web of Science ID 000325359206289

• Characterisation Of Antigens Driving In Situ Autoantibody Production In Human Lupus Tubulointerstitial Nephritis (TIN) Kinloch, A., Henderson, S., Kaverina, N., Chang, A., Haddon, D., Jarrel, J., Dunand, C., Wilson, P. C., Utz, P., Clark, M. R. WILEY-BLACKWELL. 2013: S1196

  View details for Web of Science ID 000325359206223

• 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 Balboni, I., Niewold, T. B., Morgan, G., Limb, C., Eloranta, M., Rönnblom, L., Utz, P. J., Pachman, L. M. 2013; 65 (9): 2424-2429

  Abstract

  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

• 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 Balboni, I., Niewold, T. B., Morgan, G., Limb, C., Eloranta, M., Ronnblom, L., Utz, P. J., Pachman, L. M. 2013; 65 (9): 2424-2429

  Abstract

  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 Web of Science ID 000323481400025

• Plasmid-Encoded Proinsulin Preserves C-Peptide While Specifically Reducing Proinsulin-Specific CD8+ T Cells in Type 1 Diabetes. Science translational medicine Roep, B. O., Solvason, N., Gottlieb, P. A., Abreu, J. R., Harrison, L. C., Eisenbarth, G. S., Yu, L., Leviten, M., Hagopian, W. A., Buse, J. B., von Herrath, M., Quan, J., King, R. S., Robinson, W. H., Utz, P. J., Garren, H., Steinman, L. 2013; 5 (191): 191ra82-?

  Abstract

  In type 1 diabetes (T1D), there is an intense inflammatory response that destroys the β 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 β 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-γ, 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

• Silicon-based peptide microarrays for epitope mapping of serum autoantibodies from patients with systemic lupus erythematosus with single-amino acid resolution Haddon, D., Jarrell, J., Liu, C., Price, J., Tangsombatvisit, S., Credo, G., Xu, G., Varma, M., Baechler, E., Utz, P. AMER ASSOC IMMUNOLOGISTS. 2013

  View details for Web of Science ID 000322987104099

• Influenza antigen microarrays reveal reactivity signatures associated with effective response to seasonal trivalent influenza vaccination Price, J., Jarrell, J., Furman, D., Kattah, N., Newell, E., Dekker, C., Davis, M., Utz, P. AMER ASSOC IMMUNOLOGISTS. 2013

  View details for Web of Science ID 000322987105110

• Multiplexed cytokine detection on plasmonic gold substrates with enhanced near-infrared fluorescence NANO RESEARCH Zhang, B., Price, J., Hong, G., Tabakman, S. M., Wang, H., Jarrell, J. A., Feng, J., Utz, P. J., Dai, H. 2013; 6 (2): 113-120

  View details for DOI 10.1007/s12274-012-0286-2

  View details for Web of Science ID 000314763300004

• Characterization of influenza vaccine immunogenicity using influenza antigen microarrays. PloS one Price, J. V., Jarrell, J. A., Furman, D., Kattah, N. H., Newell, E., Dekker, C. L., Davis, M. M., Utz, P. J. 2013; 8 (5)

  Abstract

  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

  View details for PubMedCentralID PMC3667171

• Characterization of influenza vaccine immunogenicity using influenza antigen microarrays. PloS one Price, J. V., Jarrell, J. A., Furman, D., Kattah, N. H., Newell, E., Dekker, C. L., Davis, M. M., Utz, P. J. 2013; 8 (5)

  View details for DOI 10.1371/journal.pone.0064555

  View details for PubMedID 23734205

• An integrated Peptide-antigen microarray on plasmonic gold films for sensitive human antibody profiling. PloS one Zhang, B., Jarrell, J. A., Price, J. V., Tabakman, S. M., Li, Y., Gong, M., Hong, G., Feng, J., Utz, P. J., Dai, H. 2013; 8 (7): e71043

  Abstract

  High-throughput screening for interactions of peptides with a variety of antibody targets could greatly facilitate proteomic analysis for epitope mapping, enzyme profiling, drug discovery and biomarker identification. Peptide microarrays are suited for such undertaking because of their high-throughput capability. However, existing peptide microarrays lack the sensitivity needed for detecting low abundance proteins or low affinity peptide-protein interactions. This work presents a new peptide microarray platform constructed on nanostructured plasmonic gold substrates capable of metal enhanced NIR fluorescence enhancement (NIR-FE) by hundreds of folds for screening peptide-antibody interactions with ultrahigh sensitivity. Further, an integrated histone peptide and whole antigen array is developed on the same plasmonic gold chip for profiling human antibodies in the sera of systemic lupus erythematosus (SLE) patients, revealing that collectively a panel of biomarkers against unmodified and post-translationally modified histone peptides and several whole antigens allow more accurate differentiation of SLE patients from healthy individuals than profiling biomarkers against peptides or whole antigens alone.

  View details for DOI 10.1371/journal.pone.0071043

  View details for PubMedID 23923050

  View details for PubMedCentralID PMC3726620

• Apoptosis and other immune biomarkers predict influenza vaccine responsiveness. Molecular systems biology Furman, D., Jojic, V., Kidd, B., Shen-Orr, S., Price, J., Jarrell, J., Tse, T., Huang, H., Lund, P., Maecker, H. T., Utz, P. J., Dekker, C. L., Koller, D., Davis, M. M. 2013; 9: 659-?

  View details for DOI 10.1038/msb.2013.15

  View details for PubMedID 23591775

• Apoptosis and other immune biomarkers predict influenza vaccine responsiveness. Molecular systems biology Furman, D., Jojic, V., Kidd, B., Shen-Orr, S., Price, J., Jarrell, J., Tse, T., Huang, H., Lund, P., Maecker, H. T., Utz, P. J., Dekker, C. L., Koller, D., Davis, M. M. 2013; 9: 659-?

  Abstract

  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

  View details for PubMedCentralID PMC3658270

• Using the allergic immune system to target cancer: activity of IgE antibodies specific for human CD20 and MUC1 CANCER IMMUNOLOGY IMMUNOTHERAPY Teo, P. Z., Utz, P. J., Mollick, J. A. 2012; 61 (12): 2295-2309

  Abstract

  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 hFcεRI 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 hFcεRI 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

• Redirecting cell-type specific cytokine responses with engineered interleukin-4 superkines NATURE CHEMICAL BIOLOGY Junttila, I. S., Creusot, R. J., Moraga, I., Bates, D. L., Wong, M. T., Alonso, M. N., Suhoski, M. M., Lupardus, P., Meier-Schellersheim, M., Engleman, E. G., Utz, P. J., Fathman, C. G., Paul, W. E., Garcia, K. C. 2012; 8 (12): 990-998

  Abstract

  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 (γc or IL-13Rα1), 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 γc was three- to ten-fold more potent than wild-type IL-4. Conversely, a variant with high affinity for IL-13Rα1 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

  View details for PubMedCentralID PMC3508151

• On silico peptide microarrays for high-resolution mapping of antibody epitopes and diverse protein-protein interactions NATURE MEDICINE Price, J. V., Tangsombatvisit, S., Xu, G., Yu, J., Levy, D., Baechler, E. C., Gozani, O., Varma, M., Utz, P. J., Liu, C. L. 2012; 18 (9): 1434-?

  Abstract

  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

  View details for PubMedCentralID PMC3491111

• New tools for classification and monitoring of autoimmune diseases NATURE REVIEWS RHEUMATOLOGY Maecker, H. T., Lindstrom, T. M., Robinson, W. H., Utz, P. J., Hale, M., Boyd, S. D., Shen-Orr, S. S., Fathman, C. G. 2012; 8 (6): 317-328

  Abstract

  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

  View details for PubMedCentralID PMC3409841

• Immune Inhibitory Molecules LAG-3 and PD-1 Synergistically Regulate T-cell Function to Promote Tumoral Immune Escape CANCER RESEARCH Woo, S., Turnis, M. E., Goldberg, M. V., Bankoti, J., Selby, M., Nirschl, C. J., Bettini, M. L., Gravano, D. M., Vogel, P., Liu, C. L., Tangsombatvisit, S., Grosso, J. F., Netto, G., Smeltzer, M. P., Chaux, A., Utz, P. J., Workman, C. J., Pardoll, D. M., Korman, A. J., Drake, C. G., Vignali, D. A. 2012; 72 (4): 917-927

  Abstract

  Inhibitory receptors on immune cells are pivotal regulators of immune escape in cancer. Among these inhibitory receptors, CTLA-4 (targeted clinically by ipilimumab) serves as a dominant off-switch while other receptors such as PD-1 and LAG-3 seem to serve more subtle rheostat functions. However, the extent of synergy and cooperative interactions between inhibitory pathways in cancer remain largely unexplored. Here, we reveal extensive coexpression of PD-1 and LAG-3 on tumor-infiltrating CD4(+) and CD8(+) T cells in three distinct transplantable tumors. Dual anti-LAG-3/anti-PD-1 antibody treatment cured most mice of established tumors that were largely resistant to single antibody treatment. Despite minimal immunopathologic sequelae in PD-1 and LAG-3 single knockout mice, dual knockout mice abrogated self-tolerance with resultant autoimmune infiltrates in multiple organs, leading to eventual lethality. However, Lag3(-/-)Pdcd1(-/-) mice showed markedly increased survival from and clearance of multiple transplantable tumors. Together, these results define a strong synergy between the PD-1 and LAG-3 inhibitory pathways in tolerance to both self and tumor antigens. In addition, they argue strongly that dual blockade of these molecules represents a promising combinatorial strategy for cancer.

  View details for DOI 10.1158/0008-5472.CAN-11-1620

  View details for Web of Science ID 000300629100011

  View details for PubMedID 22186141

  View details for PubMedCentralID PMC3288154

• Differential mTOR and ERK pathway utilization by effector CD4 T cells suggests combinatorial drug therapy of arthritis CLINICAL IMMUNOLOGY Lin, J. T., Stein, E. A., Wong, M. T., Kalpathy, K. J., Su, L. L., Utz, P. J., Robinson, W. H., Fathnnan, C. G. 2012; 142 (2): 127-138

  Abstract

  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

  View details for PubMedCentralID PMC3273543

• Correction: Specific post-translational histone modifications of neutrophil extracellular traps as immunogens and potential targets of lupus autoantibodies. Arthritis research & therapy Liu, C. L., Tangsombatvisit, S., Rosenberg, J. M., Mandelbaum, G., Gillespie, E. C., Gozani, O. P., Alizadeh, A. A., Utz, P. J. 2012; 14 (4): 403-?

  View details for DOI 10.1186/ar3933

  View details for PubMedID 22894771

  View details for PubMedCentralID PMC3580574

• Specific post-translational histone modifications of neutrophil extracellular traps as immunogens and potential targets of lupus autoantibodies (vol 14, R25, 2012) ARTHRITIS RESEARCH & THERAPY Liu, C. L., Tangsombatvisit, S., Rosenberg, J. M., Mandelbaum, G., Gillespie, E. C., Gozani, O. P., Alizadeh, A. A., Utz, P. J. 2012; 14 (4)

  View details for DOI 10.1186/ar3933

  View details for Web of Science ID 000314974600049

• Therapeutic Toll-like receptor agonists directly influence mouse and human T cell lymphoma cell viability and cytokine secretion LEUKEMIA & LYMPHOMA Landrigan, A., Yiu, G., Agarwal, K., Utz, P. J. 2012; 53 (1): 166-168

  View details for DOI 10.3109/10428194.2011.606944

  View details for Web of Science ID 000298750700029

  View details for PubMedID 21780996

  View details for PubMedCentralID PMC3339659

• Specific post-translational histone modifications of neutrophil extracellular traps as immunogens and potential targets of lupus autoantibodies ARTHRITIS RESEARCH & THERAPY Liu, C. L., Tangsombatvisit, S., Rosenberg, J. M., Mandelbaum, G., Gillespie, E. C., Gozani, O. P., Alizadeh, A. A., Utz, P. J. 2012; 14 (1)

  Abstract

  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

  View details for PubMedCentralID PMC3392818

• A proteomic approach for the identification of novel lysine methyltransferase substrates EPIGENETICS & CHROMATIN Levy, D., Liu, C. L., Yang, Z., Newman, A. M., Alizadeh, A. A., Utz, P. J., Gozani, O. 2011; 4

  Abstract

  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

  View details for PubMedCentralID PMC3212905

• T(H)1, T(H)2, and T(H)17 cells instruct monocytes to differentiate into specialized dendritic cell subsets BLOOD Alonso, M. N., Wong, M. T., Zhang, A. L., Winer, D., Suhoski, M. M., Tolentino, L. L., Gaitan, J., Davidson, M. G., Kung, T. H., Galel, D. M., Nadeau, K. C., Kim, J., Utz, P. J., Soederstroem, K., Engleman, E. G. 2011; 118 (12): 3311-3320

  Abstract

  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

  View details for PubMedCentralID PMC3179399

• CpG and Non-CpG Oligodeoxynucleotides Directly Costimulate Mouse and Human CD4(+) T Cells through a TLR9-and MyD88-Independent Mechanism JOURNAL OF IMMUNOLOGY Landrigan, A., Wong, M. T., Utz, P. J. 2011; 187 (6): 3033-3043

  Abstract

  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

  View details for PubMedCentralID PMC3169723

• Plasmonic substrates for multiplexed protein microarrays with femtomolar sensitivity and broad dynamic range NATURE COMMUNICATIONS Tabakman, S. M., Lau, L., Robinson, J. T., Price, J., Sherlock, S. P., Wang, H., Zhang, B., Chen, Z., Tangsombatvisit, S., Jarrell, J. A., Utz, P. J., Dai, H. 2011; 2

  Abstract

  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

  View details for PubMedCentralID PMC3402035

• SCREENING CyTOF-the next generation of cell detection NATURE REVIEWS RHEUMATOLOGY Cheung, R. K., Utz, P. J. 2011; 7 (9): 502-503

  View details for DOI 10.1038/nrrheum.2011.110

  View details for Web of Science ID 000294449900003

  View details for PubMedID 21788983

  View details for PubMedCentralID PMC3387986

• Reactivity profiles of broadly neutralizing anti-HIV-1 antibodies are distinct from those of pathogenic autoantibodies AIDS Singh, H., Henry, K. A., Wu, S. S., Chruscinski, A., Utz, P. J., Scott, J. K. 2011; 25 (10): 1247-1257

  Abstract

  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

  View details for PubMedCentralID PMC3334283

• Natural Killer Cells From Children With Type 1 Diabetes Have Defects in NKG2D-Dependent Function and Signaling DIABETES Qin, H., Lee, I., Panagiotopoulos, C., Wang, X., Chu, A. D., Utz, P. J., Priatel, J. J., Tan, R. 2011; 60 (3): 857-866

  Abstract

  Natural killer (NK) cells from NOD mice have numeric and functional abnormalities, and restoration of NK cell function prevents autoimmune diabetes in NOD mice. However, little is known about the number and function of NK cells in humans affected by type 1 diabetes. Therefore, we evaluated the phenotype and function of NK cells in a large cohort of type 1 diabetic children.Peripheral blood mononuclear blood cells were obtained from subjects whose duration of disease was between 6 months and 2 years. NK cells were characterized by flow cytometry, enzyme-linked immunosorbent spot assays, and cytotoxicity assays. Signaling through the activating NK cell receptor, NKG2D, was assessed by immunoblotting and reverse-phase phosphoprotein lysate microarray.NK cells from type 1 diabetic subjects were present at reduced cell numbers compared with age-matched, nondiabetic control subjects and had diminished responses to the cytokines interleukin (IL)-2 and IL-15. Analysis before and after IL-2 stimulation revealed that unlike NK cells from nondiabetic control subjects, NK cells from type 1 diabetic subjects failed to downregulate the NKG2D ligands, major histocompatibility complex class I-related chains A and B, upon activation. Moreover, type 1 diabetic NK cells also exhibited decreased NKG2D-dependent cytotoxicity and interferon-γ secretion. Finally, type 1 diabetic NK cells showed clear defects in NKG2D-mediated activation of the phosphoinositide 3-kinase-AKT pathway.These results are the first to demonstrate that type 1 diabetic subjects have aberrant signaling through the NKG2D receptor and suggest that NK cell dysfunction contributes to the autoimmune pathogenesis of type 1 diabetes.

  View details for DOI 10.2337/db09-1706

  View details for Web of Science ID 000288060300020

  View details for PubMedID 21270236

  View details for PubMedCentralID PMC3046846

• 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 NATURE IMMUNOLOGY Levy, D., Kuo, A. J., Chang, Y., Schaefer, U., Kitson, C., Cheung, P., Espejo, A., Zee, B. M., Liu, C. L., Tangsombatvisit, S., Tennen, R. I., Kuo, A. Y., Tanjing, S., Cheung, R., Chua, K. F., Utz, P. J., Shi, X., Prinjha, R. K., Lee, K., Garcia, B. A., Bedford, M. T., Tarakhovsky, A., Cheng, X., Gozani, O. 2011; 12 (1): 29-U47

  Abstract

  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-κB 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-κB-activation-linked phosphorylation of RelA at Ser311 by protein kinase C-ζ (PKC-ζ) 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

  View details for PubMedCentralID PMC3074206

• Identification of a Putative Regulator of Early T Cell Activation Genes (Reprinted from AAAS, vol 241, pg 202-205, 1988) JOURNAL OF IMMUNOLOGY Shaw, J., Utz, P. J., Durand, D. B., Toole, J., Emmel, E., Crabtree, G. R. 2010; 185 (9): 4972–75

  View details for DOI 10.1126/science.3260404

  View details for Web of Science ID 000283248700006

  View details for PubMedID 20962265

• The Ubiquitin Modifying Enzyme A20 Restricts B Cell Survival and Prevents Autoimmunity IMMUNITY Tavares, R. M., Turer, E. E., Liu, C. L., Advincula, R., Scapini, P., Rhee, L., Barrera, J., Lowel, C. A., Utz, P. J., Malynn, B. A., Ma, A. 2010; 33 (2): 181-191

  Abstract

  A20 is a ubiquitin modifying enzyme that restricts NF-kappaB signals and protects cells against tumor necrosis factor (TNF)-induced programmed cell death. Given recent data linking A20 (TNFAIP3) with human B cell lymphomas and systemic lupus erythematosus (SLE), we have generated mice bearing a floxed allele of Tnfaip3 to interrogate A20's roles in regulating B cell functions. A20-deficient B cells are hyperresponsive to multiple stimuli and display exaggerated NF-kappaB responses to CD40-induced signals. Mice expressing absent or hypomorphic amounts of A20 in B cells possess elevated numbers of germinal center B cells, autoantibodies, and glomerular immunoglobulin deposits. A20-deficient B cells are resistant to Fas-mediated cell death, probably due to increased expression of NF-kappaB-dependent antiapoptotic proteins such as Bcl-x. These findings show that A20 can restrict B cell survival, whereas A20 protects other cells from TNF-induced cell death. Our studies demonstrate how reduced A20 expression predisposes to autoimmunity.

  View details for DOI 10.1016/j.immuni.2010.07.017

  View details for Web of Science ID 000281654900008

  View details for PubMedID 20705491

  View details for PubMedCentralID PMC2931361

• Regulation of human Th9 differentiation by type I interferons and IL-21 IMMUNOLOGY AND CELL BIOLOGY Wong, M. T., Ye, J. J., Alonso, M. N., Landrigan, A., Cheung, R. K., Engleman, E., Utz, P. J. 2010; 88 (6): 624-631

  Abstract

  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

  View details for PubMedCentralID PMC3090036

• Treatment with a Toll-like receptor inhibitory GpG oligonucleotide delays and attenuates lupus nephritis in NZB/W mice AUTOIMMUNITY Graham, K. L., Lee, L. Y., Higgins, J. P., Steinman, L., Utz, P. J., Ho, P. P. 2010; 43 (2): 140-155

  Abstract

  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

  View details for PubMedCentralID PMC4150612

• IFN Regulatory Factor 5 Is Required for Disease Development in the Fc gamma RIIB(-/-)Yaa and Fc gamma RIIB-/- Mouse Models of Systemic Lupus Erythematosus JOURNAL OF IMMUNOLOGY Richez, C., Yasuda, K., Bonegio, R. G., Watkins, A. A., Aprahamian, T., Busto, P., Richards, R. J., Liu, C. L., Cheung, R., Utz, P. J., Marshak-Rothstein, A., Rifkin, I. R. 2010; 184 (2): 796-806

  Abstract

  Polymorphisms in the transcription factor IFN regulatory factor 5 (IRF5) are strongly associated in human genetic studies with an increased risk of developing the autoimmune disease systemic lupus erythematosus. However, the biological role of IRF5 in lupus pathogenesis has not previously been tested in an animal model. In this study, we show that IRF5 is absolutely required for disease development in the FcgammaRIIB(-/-)Yaa and FcgammaRIIB(-/-) lupus models. In contrast to IRF5-sufficient FcgammaRIIB(-/-)Yaa mice, IRF5-deficient FcgammaRIIB(-/-)Yaa mice do not develop lupus manifestations and have a phenotype comparable to wild-type mice. Strikingly, full expression of IRF5 is required for the development of autoimmunity, as IRF5 heterozygotes had dramatically reduced disease. One effect of IRF5 is to induce the production of the type I IFN, IFN-alpha, a cytokine implicated in lupus pathogenesis. To address the mechanism by which IRF5 promotes disease, we evaluated FcgammaRIIB(-/-)Yaa mice lacking the type I IFN receptor subunit 1. Unlike the IRF5-deficient and IRF5-heterozygous FcgammaRIIB(-/-)Yaa mice, type I IFN receptor subunit 1-deficient FcgammaRIIB(-/-)Yaa mice maintained a substantial level of residual disease. Furthermore, in FcgammaRIIB(-/-) mice lacking Yaa, IRF5-deficiency also markedly reduced disease manifestations, indicating that the beneficial effects of IRF5 deficiency in FcgammaRIIB(-/-)Yaa mice are not due only to inhibition of the enhanced TLR7 signaling associated with the Yaa mutation. Overall, we demonstrate that IRF5 plays an essential role in lupus pathogenesis in murine models and that this is mediated through pathways beyond that of type I IFN production.

  View details for DOI 10.4049/jimmunol.0901748

  View details for Web of Science ID 000273447100030

  View details for PubMedID 20007534

  View details for PubMedCentralID PMC2858062

• Targeting mTOR and MAPK Pathways to Inhibit Naive and Experienced Effector CD4 T Cells in Autoimmunity 10th Annual Meeting of the Federation-of-Clinical-Immunology-Societies Lin, J., Stein, E., Wong, M., Kalpathy, K., Su, L., Utz, P., Robinson, W., Fathman, C. ACADEMIC PRESS INC ELSEVIER SCIENCE. 2010: S69–S69

  View details for DOI 10.1016/j.clim.2010.03.211

  View details for Web of Science ID 000277953700195

• Microarray and CyTOF Characterization of Plasmacytoid Dendritic Cell Subsets Cheung, R., Goltsev, Y., Bendall, S., Nolan, G., Utz, P. J. ACADEMIC PRESS INC ELSEVIER SCIENCE. 2010: S66

  View details for DOI 10.1016/j.clim.2010.03.201

  View details for Web of Science ID 000277953700185

• Antigen Specific T Cells in Lupus Recognize U1-70 and Produce IL-17 by Tetramer Staining 10th Annual Meeting of the Federation-of-Clinical-Immunology-Societies Kattah, N., Newell, E., Chu, A., Kattah, M., Davis, M., Utz, P. ACADEMIC PRESS INC ELSEVIER SCIENCE. 2010: S9–S9

  View details for DOI 10.1016/j.clim.2010.03.033

  View details for Web of Science ID 000277953700017

• The U1-snRNP complex: structural properties relating to autoimmune pathogenesis in rheumatic diseases IMMUNOLOGICAL REVIEWS Kattah, N. H., Kattah, M. G., Utz, P. J. 2010; 233: 126-145

  Abstract

  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

  View details for PubMedCentralID PMC3074261

• Cholinergic Modulation of Angiogenesis: Role of the 7 Nicotinic Acetylcholine Receptor JOURNAL OF CELLULAR BIOCHEMISTRY Wu, J. C., Chruscinski, A., Perez, V. A., Singh, H., Pitsiouni, M., Rabinovitch, M., Utz, P. J., Cooke, J. P. 2009; 108 (2): 433-446

  Abstract

  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

  View details for PubMedCentralID PMC3140170

• Naive CD4 T Cell Proliferation Is Controlled by Mammalian Target of Rapainaycin Regulation of GRAIL Expression JOURNAL OF IMMUNOLOGY Lin, J. T., Lineberry, N. B., Kattah, M. G., Su, L. L., Utz, P. J., Fathman, C. G., Wu, L. 2009; 182 (10): 5919-5928

  Abstract

  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

  View details for PubMedCentralID PMC2853371

• Regulation of GRAIL Expression by mTOR Controls Naive CD4 T Cell Proliferation 9th Annual Meeting of the Federation-of-Clinical-Immunology-Societies Lin, J., Lineberry, N., Kattah, M., Su, L., Utz, P., Fathman, C. G. ACADEMIC PRESS INC ELSEVIER SCIENCE. 2009: S35–S36

  View details for DOI 10.1016/j.clim.2009.03.100

  View details for Web of Science ID 000266342300095

• 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 Thibault, D. L., Graham, K. L., Lee, L. Y., Balboni, I., Hertzog, P. J., Utz, P. J. 2009; 11 (4)

  Abstract

  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

  View details for PubMedCentralID PMC2745794

• Concordance of Serologic Data in Juvenile Dermatomyositis: Interferon-Alpha Induction and Activity, and Detectable Levels of Anti-Ro and Anti-La Autoantibodies Balboni, I., Niewold, T., Morgan, G., Limb, C., Eloranta, M., Utz, P., Roennblom, L., Pachman, L. ACADEMIC PRESS INC ELSEVIER SCIENCE. 2009: S134

  View details for DOI 10.1016/j.clim.2009.03.394

  View details for Web of Science ID 000266342300386

• HIT: a versatile proteomics platform for multianalyte phenotyping of cytokines, intracellular proteins and surface molecules NATURE MEDICINE Kattah, M. G., Coller, J., Cheung, R. K., Oshidary, N., Utz, P. J. 2008; 14 (11): 1284-1289

  Abstract

  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

  View details for PubMedCentralID PMC3334282

• Protein microarrays with carbon nanotubes as multicolor Raman labels NATURE BIOTECHNOLOGY Chen, Z., Tabakman, S. M., Goodwin, A. P., Kattah, M. G., Daranciang, D., Wang, X., Zhang, G., Li, X., Liu, Z., Utz, P. J., Jiang, K., Fan, S., Dai, H. 2008; 26 (11): 1285-1292

  Abstract

  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

• Expression-based Pathway Signature Analysis (EPSA): Mining publicly available microarray data for insight into human disease BMC MEDICAL GENOMICS Tenenbaum, J. D., Walker, M. G., Utz, P. J., Butte, A. J. 2008; 1

  Abstract

  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

  View details for PubMedCentralID PMC2588448

• Modulation of Peripheral B Cell Tolerance by CD72 in a Murine Model ARTHRITIS AND RHEUMATISM Li, D. H., Winslow, M. M., Cao, T. M., Chen, A. H., Davis, C. R., Mellins, E. D., Utz, P. J., Crabtree, G. R., Parnes, J. R. 2008; 58 (10): 3192-3204

  Abstract

  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

  View details for PubMedCentralID PMC2790383

• Cerebrospinal fluid cell phenotypic analysis from a phase 2 trial of the BHT-3009 DNA vaccine for multiple sclerosis Nadj, C., Nadj, I., Havrdova, E., Krasulova, E., Selmaj, K., Losy, J., Radue, E., Gianettoni, J., Solvason, N., Tersini, K., Valone, F., Utz, P. J., Robinson, W. H., Steinman, L., Garren, H., BHT 3009 Study Grp SAGE PUBLICATIONS LTD. 2008: S54

  View details for Web of Science ID 000259675700161

• Longitudinal comparison of B lymphocyte function from pristane-treated wild-type and IFNAR2-/- mice 72nd Annual Scientific Meeting of the American-College-of-Rheumatology/43rd Annual Scientific Meeting of the Association-of-Rheumatology-Health-Professionals Chu, A. D., Thibault, D. L., Utz, P. J. WILEY-BLACKWELL. 2008: S316–S317

  View details for Web of Science ID 000259244200422

• Evaluation of microarray surfaces and arraying parameters for autoantibody profiling PROTEOMICS Balboni, I., Limb, C., Tenenbaum, J. D., Utz, P. J. 2008; 8 (17): 3443-3449

  Abstract

  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

  View details for PubMedCentralID PMC3335271

• Phase 2 follow-up results of the BHT-3009 DNA vaccine for multiple sclerosis Havrdova, E., Krasulova, E., Nadj, C. G., Selmaj, K., Losy, J., Nadj, I., Radue, E., Gianettoni, J., Valone, F., Utz, P. J., Robinson, W. H., Steinman, L., Garren, H., BHT 3009 Study Grp SAGE PUBLICATIONS LTD. 2008: S47

  View details for Web of Science ID 000259675700138

• Evaluation of serum Autoantibodies from a cohort of pediatric systemic lupus erythematosus patients using autoantigen Microarray technology Balboni, I., Limb, C., Sandborg, C. I., Utz, P. J. WILEY-LISS. 2008: S502

  View details for Web of Science ID 000259244201167

• Failure of oral atorvastatin to modulate a murine model of systemic lupus erythematosus ARTHRITIS AND RHEUMATISM Graham, K. L., Lee, L. Y., Higgins, J. P., Steinman, L., Utz, P. J., Ho, P. P. 2008; 58 (7): 2098-2104

  Abstract

  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

  View details for PubMedCentralID PMC4143381

• Scalable signaling mediated by T cell antigen receptor-CD3 ITAMs ensures effective negative selection and prevents autoimmunity NATURE IMMUNOLOGY Holst, J., Wang, H., Eder, K. D., Workman, C. J., Boyd, K. L., Baquet, Z., Singh, H., Forbes, K., Chruscinski, A., Smeyne, R., van Oers, N. S., Utz, P. J., Vignali, D. A. 2008; 9 (6): 658-666

  Abstract

  The T cell antigen receptor (TCR)-CD3 complex is unique in having ten cytoplasmic immunoreceptor tyrosine-based activation motifs (ITAMs). The physiological importance of this high TCR ITAM number is unclear. Here we generated 25 groups of mice expressing various combinations of wild-type and mutant ITAMs in TCR-CD3 complexes. Mice with fewer than seven wild-type CD3 ITAMs developed a lethal, multiorgan autoimmune disease caused by a breakdown in central rather than peripheral tolerance. Although there was a linear correlation between the number of wild-type CD3 ITAMs and T cell proliferation, cytokine production was unaffected by ITAM number. Thus, high ITAM number provides scalable signaling that can modulate proliferation yet ensure effective negative selection and prevention of autoimmunity.

  View details for DOI 10.1038/ni.1611

  View details for Web of Science ID 000256067900016

  View details for PubMedID 18469818

• Protein microarrays address the elephant in the room CLINICAL CHEMISTRY Kattah, M. G., Utz, P. J., Balboni, I. 2008; 54 (6): 937-939

  View details for DOI 10.1373/clinchem.2008.104067

  View details for Web of Science ID 000256325800001

  View details for PubMedID 18509011

• Cytokines secreted in response to toll-like receptor ligand stimulation modulate differentiation of human Th17 cells ARTHRITIS AND RHEUMATISM Kattah, M. G., Wong, M. T., Yocum, M. D., Utz, P. J. 2008; 58 (6): 1619-1629

  Abstract

  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

• Peptide-coated nanotube-based biosensor for the detection of disease-specific autoantibodies in human serum BIOSENSORS & BIOELECTRONICS Drouvalakis, K. A., Bangsaruntip, S., Hueber, W., Kozar, L. G., Utz, P. J., Dai, H. 2008; 23 (10): 1413-1421

  Abstract

  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

  View details for PubMedCentralID PMC3418051

• Phase 2 trial of a DNA vaccine encoding myelin basic protein for multiple sclerosis ANNALS OF NEUROLOGY Garren, H., Robinson, W. H., Krasulova, E., Havrdova, E., Nadj, C., Selmaj, K., Losy, J., Nadj, I., Radue, E., Kidd, B. A., Gianettoni, J., Tersini, K., Utz, P. J., Valone, F., Steinman, L. 2008; 63 (5): 611-620

  Abstract

  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 Thibault, D. L., Chu, A. D., Graham, K. L., Balboni, I., Lee, L. Y., Kohlmoos, C., Landrigan, A., Higgins, J. P., Tibshirani, R., Utz, P. J. 2008; 118 (4): 1417-1426

  Abstract

  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

  View details for PubMedCentralID PMC2267033

• Toll-like receptor ligands activate mouse T cell lymphoma lines and modulate activation of primary T cells Landrigan, A., Agarwal, K., Utz, P. ACADEMIC PRESS INC ELSEVIER SCIENCE. 2008: S145

  View details for DOI 10.1016/j.clim.2008.03.417

  View details for Web of Science ID 000255533200421

• Profiling epigenetic biomarkers for the study and treatment of lupus 8th Annual Meeting of the Federation-of-Clinical-Immunology-Societies Liu, C. L., Kattah, M., Kuo, A., Shi, X., Balboni, I., Utz, P., Ozani, O. ACADEMIC PRESS INC ELSEVIER SCIENCE. 2008: S84–S84

  View details for DOI 10.1016/j.clim.2008.03.233

  View details for Web of Science ID 000255533200238

• Using the allergic immune system to target cancer: Cell activation by tumor specific IgE monoclonal antibodies 8th Annual Meeting of the Federation-of-Clinical-Immunology-Societies Teo, P., Utz, P., Mollick, J. ACADEMIC PRESS INC ELSEVIER SCIENCE. 2008: S28–S28

  View details for DOI 10.1016/j.clim.2008.03.465

  View details for Web of Science ID 000255533200077

• Evaluation of microarray surfaces and arraying parameters for autoantibody profiling Balboni, I., Limb, C., Tenenbaum, J., Utz, P. ACADEMIC PRESS INC ELSEVIER SCIENCE. 2008: S87

  View details for DOI 10.1016/j.clim.2008.03.242

  View details for Web of Science ID 000255533200247

• HIT: A novel protein array platform identifies down-regulation of CD26 on regulatory T cell subsets Kattah, M., Colter, J., Cheung, R., Oshidary, N., Utz, P. ACADEMIC PRESS INC ELSEVIER SCIENCE. 2008: S111–S112

  View details for DOI 10.1016/j.clim.2008.03.316

  View details for Web of Science ID 000255533200321

• Cytokines secreted in response to TLR-ligand stimulation modulate human Th17 differentiation Kattah, M., Wong, M., Yocum, M., Utz, P. ACADEMIC PRESS INC ELSEVIER SCIENCE. 2008: S18

  View details for DOI 10.1016/j.clim.2008.03.046

  View details for Web of Science ID 000255533200047

• Autoantigen arrays compare reactivity profiles of broadly-neutralizing HIV-1 monoclonal antibodies and Antiphospholipid syndrome (APS) antibodies 8th Annual Meeting of the Federation-of-Clinical-Immunology-Societies Singh, H., Sandhu, N., Gupta, R., Mai, M., Chruscinski, A., Utz, P., Scott, J. ACADEMIC PRESS INC ELSEVIER SCIENCE. 2008: S129–S129

  View details for DOI 10.1016/j.clim.2008.03.368

  View details for Web of Science ID 000255533200372

• Peptide-MHC arrays to detect autoreactive T cell reactivities in systemic lupus erythematosus Hanick, N., Davis, M., Utz, P. ACADEMIC PRESS INC ELSEVIER SCIENCE. 2008: S86

  View details for DOI 10.1016/j.clim.2008.03.240

  View details for Web of Science ID 000255533200245

• RAG2 PHD finger couples histone H3 lysine 4 trimethylation with V(D)J recombination NATURE Matthews, A. G., Kuo, A. J., Ramon-Maiques, S., Han, S., Champagne, K. S., Ivanov, D., Gallardo, M., Carney, D., Cheung, P., Ciccone, D. N., Walter, K. L., Utz, P. J., Shi, Y., Kutateladze, T. G., Yang, W., Gozani, O., Oettinger, M. A. 2007; 450 (7172): 1106-U18

  Abstract

  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

  View details for PubMedCentralID PMC2988437

• Free-solution oligonucleotide separation in nanoscale channels ANALYTICAL CHEMISTRY Pennathur, S., Baldessari, F., Santiago, J. G., Kattah, M. G., Steinman, J. B., Utz, P. J. 2007; 79 (21): 8316-8322

  Abstract

  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 Bar-Or, A., Vollmer, T., Antel, J., Arnold, D. L., Bodner, C. A., Campagnolo, D., Gianettoni, J., Jalili, F., Kachuck, N., Lapierre, Y., Niino, M., Oger, J., Price, M., Rhodes, S., Robinson, W. H., Shi, F., Utz, P. J., Valone, F., Weiner, L., Steinman, L., Garren, H. 2007; 64 (10): 1407-1415

  Abstract

  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 BLOOD Chan, S. M., Weng, A. P., Tibshirani, R., Aster, J. C., Utz, P. J. 2007; 110 (1): 278-286

  Abstract

  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

  View details for PubMedCentralID PMC1896117

• Technology Insight: can autoantibody profiling improve clinical practice? NATURE CLINICAL PRACTICE RHEUMATOLOGY Sharp, V., Utz, P. J. 2007; 3 (2): 96-103

  Abstract

  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 Shi, X., Kachirskaia, I., Walter, K. L., Kuo, J. A., Lake, A., Davrazou, F., Chan, S. M., Martin, D. G., Fingerman, I. M., Briggs, S. D., Howe, L., Utz, P. J., Kutateladze, T. G., Lugovskoy, A. A., Bedford, M. T., Gozani, O. 2007; 282 (4): 2450-2455

  Abstract

  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

  View details for PubMedCentralID PMC2735445

• Reverse phase protein lysate microarrays in the analysis of activated B lymphocytes 7th Annual Meeting of the Federation-of-Clinical-Immunology-Societies Chu, A. D., Chruscinski, A. J., Singh, H., Utz, P. J. ACADEMIC PRESS INC ELSEVIER SCIENCE. 2007: S84–S84

  View details for DOI 10.1016/j.clim.2007.03.417

  View details for Web of Science ID 000247137200218

• Role of MHC-linked genes in autoantigen selection and renal disease in a murine model of systemic lupus erythematosus JOURNAL OF IMMUNOLOGY Sekine, H., Graham, K. L., Zhao, S., Elliott, M. K., Ruiz, P., Utz, P. J., Gilkeson, G. S. 2006; 177 (10): 7423-7434

  Abstract

  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 PROTEOMICS Graham, K. L., Vaysberg, M., Kuo, A., Utz, P. J. 2006; 6 (21): 5720-5724

  Abstract

  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 Paniagua, R. T., Sharpe, O., Ho, P. P., Chan, S. M., Chang, A., Higgins, J. P., Tomooka, B. H., Thomas, F. M., Song, J. J., Goodman, S. B., Lee, D. M., Genovese, M. C., Utz, P. J., Steinman, L., Robinson, W. H. 2006; 116 (10): 2633-2642

  Abstract

  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

  View details for PubMedCentralID PMC1564430

• Selective tyrosine kinase inhibition by imatinib mesylate for the treatment of autoimmune arthritis. 70th Annual Scientific Meeting of the American-College-of-Rheumatology/41st Annual Scientific Meeting of the Association-of-Rheumatology-Health-Professionals Robinson, W. H., Paniagua, R. T., Ho, P. P., Chan, S. M., Sharpe, O., Utz, P. J., Genovese, M. C. WILEY-BLACKWELL. 2006: S356–S357

  View details for Web of Science ID 000240877201370

• A new two-color Fab labeling method for autoantigen protein microarrays NATURE METHODS Kattah, M. G., Alemi, G. R., Thibault, D. L., Balboni, I., Utz, P. J. 2006; 3 (9): 745-751

  Abstract

  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 Chan, S. M., Olson, J. A., Utz, P. J. 2006; 69 (2): 59-65

  Abstract

  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

• Single-cell analysis of siRNA-mediated gene silencing using multiparameter flow cytometry CYTOMETRY PART A Chan, S. M., Olson, J. A., Utz, P. J. 2006; 69A (2): 59-65

  Abstract

  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 DOI 10.1002/cyto.a.20209

  View details for Web of Science ID 000235181100001

• Multiplexed protein array platformsfor analysis of autoimmune disease. Annu. Rev. Immunol. Balboni I., Chan SM, Kattah M, Genenbaum JD, Butte AJ, Utz PJ. 2006; 24: 391-418
• Using the allergic immune system to target breast cancer: production of anti-MUC1 IgE monoclonal antibodies. 29th Annual San Antonio Breast Cancer Symposium Mollick, J. A., Teo, P., Utz, P. J. SPRINGER. 2006: S78–S78

  View details for Web of Science ID 000242047100226

• ELECTROPHORESIS IN NANOCHANNELS 2nd US-European Fluids Engineering Division Summer Meeting/14th International Conference on Nuclear Engineering Pennathur, S., Baldessari, F., Kattah, M., Utz, P. J., Santiago, J. G. AMER SOC MECHANICAL ENGINEERS. 2006: 589–593

  View details for Web of Science ID 000272191800082

• Role of MHC- linked genes in autoantigen selection and renal disease in a murine model of SLE. Journal of Immunology Sekine H., K.L. Graham, S. Zhao, M.K. Elliott, P. Ruiz, L. Morel, V.M. Holers, P.J. Utz, G.S. Gilkeson 2006; in press
• Autoantibody profiling of lupus mice deficient for interferon signaling components. 6th Annual Meeting of the Federation-of-Clinical-Immunology-Societies Thibault, D., Graham, K., Balboni, I., Lee, L., Kohlmoos, C., Tibshirani, R., Utz, P. ACADEMIC PRESS INC ELSEVIER SCIENCE. 2006: S72–S73

  View details for DOI 10.1016/j.clim.2006.04.102

  View details for Web of Science ID 000237924300184

• 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 Huang, M., Ida, H., KAMACHI, M., Iwanaga, N., Izumi, Y., Tanaka, F., Aratake, K., Arima, K., Tamai, M., Hida, A., Nakamura, H., Origuchi, T., Kawakami, A., Ogawa, N., Sugai, S., Utz, P. J., Eguchi, K. 2005; 142 (1): 148-154

  Abstract

  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

  View details for PubMedCentralID PMC1809481

• Antigen microarray profiling of autoantibodies in rheumatoid arthritis ARTHRITIS AND RHEUMATISM Hueber, W., Kidd, B. A., Tomooka, B. H., Lee, B. J., Bruce, B., Fries, J. F., Sonderstrup, G., Monach, P., Drijfhout, J. W., van Venrooij, W. J., Utz, P. J., Genovese, M. C., Robinson, W. H. 2005; 52 (9): 2645-2655

  Abstract

  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

• Sources of autoantigens in systemic lupus erythematosus CURRENT OPINION IN RHEUMATOLOGY Graham, K. L., Utz, P. J. 2005; 17 (5): 513-517

  Abstract

  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

• An array of possibilities for the study of autoimmunity NATURE Fathman, C. G., Soares, L., Chan, S. M., Utz, P. J. 2005; 435 (7042): 605-611

  Abstract

  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 Graham, K. L., Thibault, D. L., Steinman, J. B., Okeke, L., Kao, P. N., Utz, P. J. 2005; 52 (6): 1684-1693

  Abstract

  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 IMMUNOLOGICAL REVIEWS Utz, P. J. 2005; 204: 264-282

  Abstract

  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

• Suppression of autoimmunity via microbial mimics of altered peptide ligands MOLECULAR MIMICRY: INFECTION-INDUCING AUTOIMMUNE DISEASE Steinman, L., Utz, P. J., Robinson, W. H. 2005; 296: 55-63

  Abstract

  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) cell death. Modern rheumatology Ida, H., Utz, P. J., Anderson, P., Eguchi, K. 2005; 15 (5): 315-322

  Abstract

  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

• The challenge of analyzing the proteome in humans with autoimmune diseases Conference on Human Immunology Chan, S. M., Utz, P. J. NEW YORK ACAD SCIENCES. 2005: 61–68

  Abstract

  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

• Granzyme B and natural killer (NK) death Modern Rheumatology I. Ida, P.J. Utz, P. Anderson, K. Eguchi 2005; 15: 315-322.
• Modulation of murine lupus by an inhibitory GpG oligonucleotide 5th Annual Meeting of the Federation-of-Clinical-Immunology-Societies Graham, K. L., Lee, L. Y., Teo, P., Steinman, L., Utz, P. J., Ho, P. P. ACADEMIC PRESS INC ELSEVIER SCIENCE. 2005: S109–S110

  View details for Web of Science ID 000229104400306

• Suppression of Lck sensitizes acute lymphoblastic T cells to the antiproliferative action of interferon alpha. 5th Annual Meeting of the Federation-of-Clinical-Immunology-Societies Chan, S. M., Utz, P. J. ACADEMIC PRESS INC ELSEVIER SCIENCE. 2005: S183–S183

  View details for Web of Science ID 000229104400516

• Protein microarrays for multiplex analysis of signal transduction pathways NATURE MEDICINE Chan, S. M., Ermann, J., Su, L., Fathman, C. G., Utz, P. J. 2004; 10 (12): 1390-1396

  Abstract

  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 Su, L., Creusot, R. J., Gallo, E. M., Chan, S. M., Utz, P. J., Fathman, C. G., Ermann, J. 2004; 173 (8): 4994-5001

  Abstract

  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 Hueber, W., Zeng, D. F., Strober, S., Utz, P. J. 2004; 50 (10): 3239-3249

  Abstract

  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

• High-throughput methods for measuring autoantibodies in systemic lupus erythematosus and other autoimmune diseases AUTOIMMUNITY Graham, K. L., Robinson, W. H., Steinman, L., Utz, P. J. 2004; 37 (4): 269-272

  Abstract

  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

• Proteolytic cleavage of the catalytic subunit of DNA-dependent protein kinase during poliovirus infection JOURNAL OF VIROLOGY Graham, K. L., Gustin, K. E., RIVERA, C., Kuyumcu-Martinez, N. M., Choe, S. S., Lloyd, R. E., Sarnow, P., Utz, P. J. 2004; 78 (12): 6313-6321

  Abstract

  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

  View details for PubMedCentralID PMC416498

• Characterization of novel antigens recognized by serum autoantibodies from anti-CD1 TCR-transgenic lupus mice EUROPEAN JOURNAL OF IMMUNOLOGY Hueber, W., Zeng, D. F., Sharpe, O., Robinson, W. H., Strober, S., Utz, P. J. 2004; 34 (6): 1654-1662

  Abstract

  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

• "hot technologies" for clinical immunology research CLINICAL IMMUNOLOGY Utz, P. J. 2004; 111 (2): 153-154

  View details for Web of Science ID 000221681800001

  View details for PubMedID 15137947

• Unlocking the "PAD" lock on rheumatoid arthritis ANNALS OF THE RHEUMATIC DISEASES Utz, P. J., Genovese, M. C., Robinson, W. H. 2004; 63 (4): 330-332

  View details for DOI 10.1136/ard.2003.015990

  View details for Web of Science ID 000220198000002

  View details for PubMedID 15020322

  View details for PubMedCentralID PMC1754966

• Antibodies in scleroderma: direct pathogenicity and phenotypic associations. Current rheumatology reports Chung, L., Utz, P. J. 2004; 6 (2): 156-163

  Abstract

  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 LUPUS Utz, P. 2004; 13 (5): 304-311

  Abstract

  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 PROTEOMICS Robinson, W. H., Steinman, L., Utz, P. J. 2003; 3 (11): 2077-2084

  Abstract

  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 NATURE BIOTECHNOLOGY Robinson, W. H., Fontoura, P., Lee, B. J., de Vegvar, H. E., Tom, J., Pedotti, R., DiGennaro, C. D., Mitchell, D. J., Fong, D., Ho, P. P., Ruiz, P. J., Maverakis, E., Stevens, D. B., Bernard, C. C., Martin, R., Kuchroo, V. K., van Noort, J. M., Genain, C. P., Amor, S., Olsson, T., Utz, P. J., Garren, H., Steinman, L. 2003; 21 (9): 1033-1039

  Abstract

  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 67th Annual Scientific Meeting of the American-College-of-Rheumatology/38th Annual Scientific Meeting of the Association-of-Rheumatology-Health-Professionals Hueber, W., Lee, B. J., Genovese, M. C., Bruce, B., van Venrooij, W. J., Smolen, J. S., Steinman, L., Utz, P. J., Robinson, W. H. 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 Hueber, W., Utz, P. J., Robinson, W. H. 2003; 21 (5): S59-S64

  Abstract

  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 Chen, R. J., Bangsaruntip, S., Drouvalakis, K. A., Kam, N. W., Shim, M., Li, Y. M., Kim, W., Utz, P. J., DAI, H. J. 2003; 100 (9): 4984-4989

  Abstract

  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

  View details for PubMedCentralID PMC154284

• On-chip coupling of isoelectric focusing and free solution electrophoresis for multidimensional separations ANALYTICAL CHEMISTRY Herr, A. E., Molho, J. I., Drouvalakis, K. A., Mikkelsen, J. C., Utz, P. J., Santiago, J. G., Kenny, T. W. 2003; 75 (5): 1180-1187

  Abstract

  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

• Multiplex autoantibody profiling using 'synovial proteome' microarrays identifies citrulline-modified peptides as major targets of the autoimmune response in rheumatoid arthritis 3rd World Congress of the Global-Arthritis-Research-Network (GARN) Hueber, W., Lee, B. J., Genovese, M., van Venrooij, W., Steinman, L., Utz, P. J., Robinson, W. H. BIOMED CENTRAL LTD. 2003: S31–S31

  View details for DOI 10.1186/ar900

  View details for Web of Science ID 000220116700101

• Interpreting interest in interferon-alpha ARTHRITIS RESEARCH & THERAPY Thibault, D. L., Utz, P. J. 2003; 5 (5): 246-248

  View details for DOI 10.1186/ar796

  View details for Web of Science ID 000184729800008

  View details for PubMedID 12932285

  View details for PubMedCentralID PMC193733

• Protein and peptide array analysis of autoimmune disease BIOTECHNIQUES Robinson, W. H., Steinman, L., Utz, P. J. 2002: 66-69

  Abstract

  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 KAMACHI, M., Le, T. M., Kim, S. J., Geiger, M. E., Anderson, P., Utz, P. J. 2002; 196 (9): 1213-1225

  Abstract

  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

  View details for PubMedCentralID PMC2194102

• Millennium Award. Proteomics for the development of DNA tolerizing vaccines to treat autoimmune disease. Clinical immunology Robinson, W. H., Garren, H., Utz, P. J., Steinman, L. 2002; 103 (1): 7-12

  Abstract

  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 for the development of DNA tolerizing vaccines to treat autoimmune disease CLINICAL IMMUNOLOGY Robinson, W. H., Garren, H., Utz, P. J., Steinman, L. 2002; 103 (1): 7-12

  Abstract

  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 DOI 10.1006/clim.2002.5185

  View details for Web of Science ID 000175409000002

• Proteomics technologies for the study of autoimmune disease ARTHRITIS AND RHEUMATISM Robinson, W. H., Steinman, L., Utz, P. J. 2002; 46 (4): 885-893

  View details for Web of Science ID 000174946500006

  View details for PubMedID 11953963

• Autoantigen microarrays for multiplex characterization of autoantibody responses NATURE MEDICINE Robinson, W. H., DiGennaro, C., Hueber, W., Haab, B. B., KAMACHI, M., Dean, E. J., Fournel, S., Fong, D., Genovese, M. C., de Vegvar, H. E., Skriner, K., Hirschberg, D. L., Morris, R. I., Muller, S., Pruijn, G. J., van Venrooij, W. J., Smolen, J. S., Brown, P. O., Steinman, L., Utz, P. J. 2002; 8 (3): 295-301

  Abstract

  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

• Monoclonal antibodies derived from BALB/c mice immunized with apoptotic Jurkat T cells recognize known autoantigens JOURNAL OF AUTOIMMUNITY Gensler, T. J., Hottelet, M., Zhang, C. H., Schlossman, S., Anderson, P., Utz, P. J. 2001; 16 (1): 59-69

  Abstract

  It has been postulated that post-translational modifications and relocalization of proteins during apoptosis may lead to presentation of these molecules to the immune system in such a way that normal mechanisms of tolerance are bypassed. In the present study, Jurkat cells were induced to undergo apoptosis by treatment with the chemotherapeutic agent Ara-C. BALB/c mice were then immunized with the apoptotic cells and hybridomas were generated. Using an indirect immunofluorescence assay, the monoclonal antibodies produced were screened by flow cytometry for those monoclonal antibodies demonstrating reactivity with permeabilized apoptotic Jurkat cells but not with non-permeabilized normal or apoptotic Jurkat cells. Of 281 monoclonal antibodies, 20 monoclonal antibodies with these properties were selected for further analysis. Using 32P- or 35S-metabolically labelled Jurkat cells, these selected monoclonal antibodies were screened for their ability to recognize autoantigens by immunoprecipitation and Western blotting. Well characterized autoimmune sera were then used to confirm the identity of autoantigens by immunoblotting. We demonstrate that immunization of normal mice with apoptotic Jurkat cells results in the formation of antibodies targeting multiple autoantigens or autoantigen complexes, including Ku, rRNPs, snRNPs and vimentin. These findings are consistent with the hypothesis that apoptosis can contribute to the development of autoimmunity.

  View details for DOI 10.1006/jaut.2000.0464

  View details for Web of Science ID 000167079400007

  View details for PubMedID 11221997

• 21st European Workshop for Rheumatology Research, Vienna, Austria, 1-4 March 2001. Arthritis research Utz, P. J. 2001; 3 (4): 237-240

  Abstract

  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

  View details for PubMedCentralID PMC128901

• Life and death decisions: regulation of apoptosis by proteolysis of signaling molecules CELL DEATH AND DIFFERENTIATION Utz, P. J., Anderson, P. 2000; 7 (7): 589-602

  Abstract

  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

• Small nucleolar RNP scleroderma autoantigens associate with phosphorylated serine/arginine splicing factors during apoptosis ARTHRITIS AND RHEUMATISM Overzet, K., Gensler, T. J., Kim, S. J., Geiger, M. E., van Venrooij, W. J., Pollard, K. M., Anderson, P., Utz, P. J. 2000; 43 (6): 1327-1336

  Abstract

  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

• The fate of U1 snRNP during anti-Fas induced apoptosis: specific cleavage of the U1 snRNA molecule CELL DEATH AND DIFFERENTIATION Degen, W. G., van Aarssen, Y., Pruijn, G. J., Utz, P. J., van Venrooij, W. J. 2000; 7 (1): 70-79

  Abstract

  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 Rutjes, S. A., Utz, P. J., van der Heijden, A., Broekhuis, C., van Venrooij, W. J., Pruijn, G. J. 1999; 6 (10): 976-986

  Abstract

  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 Rutjes, S. K., van der Heijden, A., Utz, P. J., van Venrooij, W. J., Pruijn, G. J. 1999; 274 (35): 24799-24807

  Abstract

  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

• Posttranslational protein modifications, apoptosis, and the bypass of tolerance to autoantigens ARTHRITIS AND RHEUMATISM Utz, P. J., Anderson, P. 1998; 41 (7): 1152-1160

  View details for Web of Science ID 000074585400002

  View details for PubMedID 9663470

• SUP-HD1 - A NEW HODGKINS DISEASE-DERIVED CELL-LINE WITH LYMPHOID FEATURES PRODUCES INTERFERON-GAMMA BLOOD Naumovski, L., Utz, P. J., Bergstrom, S. K., Morgan, R., Molina, A., Toole, J. J., Glader, B. E., McFall, P., Weiss, L. M., Warnke, R., Smith, S. D. 1989; 74 (8): 2733-2742

  Abstract

  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