Publications by authors named "Raphael Hirsch"

36 Publications

Regulation of Pulmonary Bacterial Immunity by Follistatin-Like Protein 1.

Infect Immun 2020 Dec 15;89(1). Epub 2020 Dec 15.

Division of Pediatric Infectious Diseases, Department of Pediatrics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA

is a common cause of antibiotic-resistant pneumonia. Follistatin-like protein 1 (FSTL-1) is highly expressed in the lung and is critical for lung homeostasis. The role of FSTL-1 in immunity to bacterial pneumonia is unknown. Wild-type (WT) and FSTL-1 hypomorphic (Hypo) mice were infected with to determine infectious burden, immune cell abundance, and cytokine production. FSTL-1 Hypo/TCRδ and FSTL-1 Hypo/IL17ra were also generated to assess the role of γδT17 cells in this model. FSTL-1 Hypo mice had reduced lung burden compared with that of WT controls. FSTL-1 Hypo mice had increased /interleukin-17A (IL-17A) and IL-17-dependent cytokine expression. FSTL-1 Hypo lungs also had increased IL-17A and TCRγδ cells. FSTL-1 Hypo/TCRδ displayed a lung burden similar to that of FSTL-1 Hypo and reduced lung burden compared with the TCRδ controls. However, FSTL-1 Hypo/TCRδ mice had greater bacterial dissemination than FSTL-1 Hypo mice, suggesting that gamma delta T (γδT) cells are dispensable for FSTL-1 Hypo control of pulmonary infection but are required for dissemination control. Confusing these observations, FSTL-1 Hypo/TCRδ lungs had an increased percentage of IL-17A-producing cells compared with that of TCRδ mice. Removal of IL-17A signaling in the FSTL-1 Hypo mouse resulted in an increased lung burden. These findings identify a novel role for FSTL-1 in innate lung immunity to bacterial infection, suggesting that FSTL-1 influences type-17 pulmonary bacterial immunity.
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http://dx.doi.org/10.1128/IAI.00298-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7927939PMC
December 2020

The Follistatin-like Protein 1 Pathway Is Important for Maintaining Healthy Articular Cartilage.

ACR Open Rheumatol 2020 Jul 12;2(7):407-414. Epub 2020 Jun 12.

University of Iowa Carver College of Medicine, Iowa City.

Objective: We sought to determine whether follistatin-like protein 1 (FSTL1), a protein produced by articular chondrocytes, promotes healthy articular cartilage and prevents chondrocytes from undergoing terminal differentiation to hypertrophic cells.

Methods: In vitro experiments were performed with immortalized human articular chondrocytes. The cells were transduced with a lentivirus encoding human FSTL1 small hairpin RNA or with an adenovirus encoding FSTL1. A quantitative polymerase chain reaction was used for gene expression analysis. Protein expression was assessed by Western blotting. Co-immunoprecipitation was used to identify interacting partners of FSTL1. FSTL1 expression in human articular cartilage was analyzed using confocal microscopy.

Results: Downregulation of FSTL1 expression in transforming growth factor β (TGFβ)-stimulated chondrocyte pellet cultures led to chondrocyte terminal differentiation characterized by poor production of cartilage extracellular matrix and altered expression of genes and proteins involved in cartilage homeostasis, including MMP13, COL10A1, RUNX2, COL2A1, ACAN, Sox9, and phospho-Smad3. We also showed that FSTL1 interacts with TGFβ receptor proteins, Alk1 and endoglin, suggesting a potential mechanism for its effects on chondrocytes. Transduction of chondrocytes with an FSTL1 transgene increased COL2A1 expression, whereas it did not affect MMP13 expression. FSTL1 protein expression was decreased in human osteoarthritic cartilage in situ.

Conclusion: Our data suggest that FSTL1 plays an important role in maintaining healthy articular cartilage and the FSTL1 pathway may represent a therapeutic target for degenerative diseases of cartilage.
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http://dx.doi.org/10.1002/acr2.11155DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7368136PMC
July 2020

FSTL-1 Attenuation Causes Spontaneous Smoke-Resistant Pulmonary Emphysema.

Am J Respir Crit Care Med 2020 04;201(8):934-945

Division of Pediatric Infectious Diseases.

The role of FSTL-1 (follistatin-like 1) in lung homeostasis is unknown. We aimed to define the impact of FSTL-1 attenuation on lung structure and function and to identify FSTL-1-regulated transcriptional pathways in the lung. Further, we aimed to analyze the association of FSTL-1 SNPs with lung disease. FSTL-1 hypomorphic (FSTL-1 Hypo) mice underwent lung morphometry, pulmonary function testing, and micro-computed tomography. expression was determined in wild-type lung cell populations from three independent research groups. RNA sequencing of wild-type and FSTL-1 Hypo mice identified FSTL-1-regulated gene expression, followed by validation and mechanistic examination. SNP analysis was performed in the COPDGene (Genetic Epidemiology of Chronic Obstructive Pulmonary Disease) cohort. FSTL-1 Hypo mice developed spontaneous emphysema, independent of smoke exposure. is highly expressed in the lung by mesenchymal and endothelial cells but not immune cells. RNA sequencing of whole lung identified 33 FSTL-1-regulated genes, including , an orphan nuclear hormone receptor that negatively regulates NF-κB (nuclear factor-κB) signaling. , recombinant FSTL-1 treatment of macrophages attenuated NF-κB p65 phosphorylation in an -dependent manner. Within the COPDGene cohort, several SNPs in the region corresponded to chronic obstructive pulmonary disease and lung function. This work identifies a novel role for FSTL-1 protecting against emphysema development independent of smoke exposure. This FSTL-1-deficient emphysema implicates regulation of immune tolerance in lung macrophages through . Further study of the mechanisms involving FSTL-1 in lung homeostasis, immune regulation, and NF-κB signaling may provide additional insight into the pathophysiology of emphysema and inflammatory lung diseases.
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http://dx.doi.org/10.1164/rccm.201905-0973OCDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7159415PMC
April 2020

Expanding the Pipeline for Pediatric Physician-Scientists.

J Pediatr 2019 04;207:3-7.e1

Department of Pediatrics, Perelman School of Medicine at the University of Pennsylvania, Children's Hospital of Philadelphia, Philadelphia, PA. Electronic address:

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http://dx.doi.org/10.1016/j.jpeds.2019.01.025DOI Listing
April 2019

Follistatin-like protein 1 modulates IL-17 signaling via IL-17RC regulation in stromal cells.

Immunol Cell Biol 2017 09 5;95(8):656-665. Epub 2017 Apr 5.

Children's Hospital of Pittsburgh of UPMC, Pittsburgh, PA, USA.

Follistatin-like protein 1 (FSTL-1) possesses several newly identified roles in mammalian biology, including interleukin (IL)-17-driven inflammation, though the mechanism underlying FSTL-1 influence on IL-17-mediated cytokine production is unknown. Using parallel in vitro bone marrow stromal cell models of FSTL-1 suppression, we employed unbiased microarray analysis to identify FSTL-1-regulated genes and pathways that could influence IL-17-dependent production of IL-6 and granulocyte colony-stimulating factor. We discovered that FSTL-1 modulates Il17rc gene expression. Specifically, FSTL-1 was necessary for Il17rc gene transcription, IL-17RC surface protein expression and IL-17-dependent cytokine production. This work identifies a mechanism by which FSTL-1 influences IL-17-driven inflammatory signaling in vitro and reveals a novel function for FSTL-1, as a modulator of gene expression. Thus enhanced understanding of the interplay between FSTL-1 and IL-17-mediated inflammation may provide insight into potential therapeutic targets of IL-17-mediated diseases and warrants ongoing study of in vivo models and clinical scenarios of FSTL-1-influenced diseases.
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http://dx.doi.org/10.1038/icb.2017.26DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5609702PMC
September 2017

Suppression of arthritis-induced bone erosion by a CRAC channel antagonist.

RMD Open 2016 8;2(1):e000093. Epub 2016 Jan 8.

Department of Microbiology, Immunology & Cell Biology, and the Mary Babb Randolph Cancer Center , West Virginia University School of Medicine , Morgantown, West Virginia , USA.

Objective: We have shown in vitro and in vivo that osteoclast maturation requires calcium-release activated calcium (CRAC) channels. In inflammatory arthritis, osteoclasts mediate severe and debilitating bone erosion. In the current study, we assess the value of CRAC channels as a therapeutic target to suppress bone erosion in acute inflammatory arthritis.

Methods: Collagen-induced arthritis (CIA) was induced in mice. The CRAC channel inhibitor 3,4-dichloropropionaniline (DCPA) and a placebo was administered 1 day prior to collagen II booster to induce arthritis. Effects on swelling, inflammatory cell invasion in joints, serum cytokines and bone erosion were measured.

Results: Assays, by blinded observers, of arthritis severity showed that DCPA, 21 mg/kg/day, suppressed arthritis development over 3 weeks. Bone and cartilage damage in sections of animal feet was reduced approximately 50%; overall swelling of joints was reduced by a similar amount. Effects on bone density by µCT showed clear separation in DCPA-treated CIA animals from CIA without treatment, while differences between controls without CIA and CIA treated with DCPA differed by small amounts and in most cases were not statistically different. Response was not related to anticollagen titres. There were no adverse effects in the treated group on animal weight or activity, consistent with low toxicity. The effect was maximal 12-17 days after collagen booster, during the rapid appearance of arthritis in untreated CIA. At 20 days after treatment (day 40), differences in arthritis score were reduced and tumour necrosis factor α, interleukin (IL)-1, or IL-6 in the serum of the animals were similar in treated and untreated animals.

Conclusions: DCPA, a novel inhibitor of CRAC channels, suppresses bone erosion associated with acute arthritis in mice and might represent a new treatment modality for acute arthrits.
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http://dx.doi.org/10.1136/rmdopen-2015-000093DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4716559PMC
January 2016

Follistatin-like protein 1 and its role in inflammation and inflammatory diseases.

Immunol Res 2014 Aug;59(1-3):266-72

Stead Family Department of Pediatrics, University of Iowa Carver College of Medicine, 2191 ML, 500 Newton Road, Iowa City, IA, 52242, USA,

Follistatin-like protein 1 (FSTL1) is a secreted glycoprotein produced mainly by cells of mesenchymal origin. FSTL1 has been shown to play an important role during embryogenesis; FSTL1-deficient mice die at birth from multiple developmental abnormalities. In the last decade, FSTL1 has been identified as a novel inflammatory protein, enhancing synthesis of proinflammatory cytokines and chemokines by immune cells in vitro and in vivo. FSTL1 mediates proinflammatory events in animal models of inflammatory diseases, particularly in collagen-induced arthritis in mice. FSTL1 is elevated in various inflammatory conditions and decreased during the course of treatment. FSTL1 may therefore be a valuable biomarker for such diseases. Moreover, a variety of experiments suggest that targeting of FSTL1 may be useful in the treatment of diseases in which inflammation plays a central role.
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http://dx.doi.org/10.1007/s12026-014-8526-zDOI Listing
August 2014

Follistatin-like protein 1 is a critical mediator of experimental Lyme arthritis and the humoral response to Borrelia burgdorferi infection.

Microb Pathog 2014 Aug 24;73:70-9. Epub 2014 Apr 24.

Department of Pediatrics, University of Pittsburgh School of Medicine, 4401 Penn Avenue, Pittsburgh, PA 15224, USA. Electronic address:

Follistatin-like protein 1 (FSTL-1) has recently been described as a critical mediator of CIA and a marker of disease activity. Lyme arthritis, caused by Borrelia burgdorferi, shares similarities with autoimmune arthritis and the experimental murine model collagen-induced arthritis (CIA). Because FSTL-1 is important in CIA and autoimmune arthritides, and Lyme arthritis shares similarities with CIA, we hypothesized that FSTL-1 may be an important mediator of Lyme arthritis. We demonstrate for the first time that FSTL-1 is induced by B. burgdorferi infection and is required for the development of Lyme arthritis in a murine model, utilizing a gene insertion to generate FSTL-1 hypomorphic mice. Using qPCR and qRT-PCR, we found that despite similar early infectious burden, FSTL-1 hypomorphic mice have improved spirochetal clearance in the face of attenuated arthritis and inflammatory cytokine production. Further, FSTL-1 mediates pathogen-specific antibody production and antigen recognition when assessed by ELISA and one- and two-dimensional immunoblotting. This study is the first to describe a role for FSTL-1 in the development of Lyme arthritis and anti-Borrelia response, and the first to demonstrate a role for FSTL-1 in response to infection, highlighting the potential for FSTL-1 as a target in the treatment of B. burgdorferi infection.
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http://dx.doi.org/10.1016/j.micpath.2014.04.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4899828PMC
August 2014

Follistatin-like protein 1 regulates chondrocyte proliferation and chondrogenic differentiation of mesenchymal stem cells.

Ann Rheum Dis 2015 Jul 18;74(7):1467-73. Epub 2014 Mar 18.

Department of Pediatrics, University of Iowa Carver College of Medicine, Iowa City, Iowa, USA.

Objectives: Chondrocytes, the only cells in the articular cartilage, play a pivotal role in osteoarthritis (OA) because they are responsible for maintenance of the extracellular matrix (ECM). Follistatin-like protein 1 (FSTL1) is a secreted protein found in mesenchymal stem cells (MSCs) and cartilage but whose function is unclear. FSTL1 has been shown to modify cell growth and survival. In this work, we sought to determine whether FSTL1 could regulate chondrogenesis and chondrogenic differentiation of MSCs.

Methods: To study the role of FSTL1 in chondrogenesis, we used FSTL1 knockout (KO) mice generated in our laboratory. Proliferative capacity of MSCs, obtained from skulls of E18.5 embryos, was analysed by flow cytometry. Chondrogenic differentiation of MSCs was carried out in a pellet culture system. Gene expression differences were assessed by microarray analysis and real-time PCR. Phosphorylation of Smad3, p38 MAPK and Akt was analysed by western blotting.

Results: The homozygous FSTL1 KO embryos showed extensive skeletal defects and decreased cellularity in the vertebral cartilage. Cell proliferation of FSTL1-deficient MSCs was reduced. Gene expression analysis in FSTL1 KO MSCs revealed dysregulation of multiple genes important for chondrogenesis. Production of ECM proteoglycans and collagen II expression were decreased in FSTL1-deficient MSCs differentiated into chondrocytes. Transforming growth factor β signalling in FSTL1 KO cells was significantly suppressed.

Conclusions: FSTL1 is a potent regulator of chondrocyte proliferation, differentiation and expression of ECM molecules. Our findings may lead to the development of novel strategies for cartilage repair and provide new disease-modifying treatments for OA.
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http://dx.doi.org/10.1136/annrheumdis-2013-204822DOI Listing
July 2015

Follistatin-like protein 1 enhances NLRP3 inflammasome-mediated IL-1β secretion from monocytes and macrophages.

Eur J Immunol 2014 May 20;44(5):1467-79. Epub 2014 Feb 20.

Department of Pediatrics, University of Iowa Carver College of Medicine, Iowa City, IA, USA.

Follistatin-like protein 1 (FSTL-1) is overexpressed in a number of inflammatory conditions characterized by elevated IL-1β. Here, we found that FSTL-1 serum concentration was increased threefold in patients with bacterial sepsis and fourfold following administration of LPS to mice. To test the contribution of FSTL-1 to IL-1β secretion, WT and FSTL-1-deficient mice were injected with LPS. While LPS induced IL-1β in the sera of WT mice, it was low or undetectable in FSTL-1-deficient mice. Monocytes/macrophages, a key source of IL-1β, do not normally express FSTL-1. However, FSTL-1 was found in tissue macrophages after injection of LPS into mouse footpads, demonstrating that macrophages are capable of taking up FSTL-1 at sites of inflammation. In vitro, intracellular FSTL-1 localized to the mitochondria. FSTL-1 activated the mitochondrial electron transport chain, increased the production of ATP (a key activator of the nod-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome) and IL-1β secretion. FSTL-1 also enhanced transcription of the NLRP3 and procaspase 1 genes, two components of the NLRP3 inflammasome. Adenovirus-mediated overexpression of FSTL-1 in mouse paws led to activation of the inflammasome complex and local secretion of IL-1β and IL-1β-related proinflammatory cytokines. These results suggest that FSTL-1 may act on the NLRP3 inflammasome to promote IL-1β secretion from monocytes/macrophages.
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http://dx.doi.org/10.1002/eji.201344063DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4004659PMC
May 2014

Follistatin-like protein 1 and the ferritin/erythrocyte sedimentation rate ratio are potential biomarkers for dysregulated gene expression and macrophage activation syndrome in systemic juvenile idiopathic arthritis.

J Rheumatol 2013 Jul 15;40(7):1191-9. Epub 2013 May 15.

Division of Rheumatology, Children's Hospital of Pittsburgh, University of Pittsburgh Medical Center (UPMC), University of Pittsburgh, School of Medicine, Pittsburgh, Pennsylvania, USA.

Objective: Follistatin-like protein 1 (FSTL-1) is a secreted glycoprotein overexpressed in certain inflammatory diseases. Our objective was to correlate FSTL-1 levels with gene expression, known biomarkers, and measures of disease activity in systemic juvenile idiopathic arthritis (sJIA), including macrophage activation syndrome (MAS).

Methods: FSTL-1 serum levels were measured by ELISA in 28 patients with sJIA, including 7 patients who developed MAS, and 30 healthy controls. Levels were correlated with erythrocyte sedimentation rate (ESR), ferritin, and soluble interleukin-2 receptor-α (sIL-2Rα). Gene expression based on FSTL-1 levels was analyzed in peripheral blood mononuclear cells (PBMC).

Results: Serum levels of FSTL-1 were elevated at time of presentation of sJIA (mean 200.7 ng/ml) and decreased to normal (mean 133.7 ng/ml) over 24 months (p < 0.01). FSTL-1 levels were markedly elevated during acute MAS (mean 279.8 ng/ml) and decreased to normal following treatment (p < 0.001). FSTL-1 levels correlated with serum markers of inflammation, including sIL-2Rα and ferritin. Ferritin/ESR ratio was superior to ferritin, sIL-2Rα, and FSTL-1 in discriminating MAS from new-onset sJIA. PBMC from patients with FSTL-1 levels > 200 ng/ml showed altered expression of genes related to innate immunity, erythropoiesis, and natural killer cell dysfunction. Two patients with the highest FSTL-1 levels at disease onset (> 300 ng/ml) ultimately developed MAS.

Conclusion: Elevated pretreatment serum FSTL-1 levels in sJIA are associated with dysregulated gene expression suggestive of occult MAS, and may have utility in predicting progression to overt MAS. Ferritin/ESR ratio may be superior to ferritin alone in discriminating overt MAS from new-onset sJIA.
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http://dx.doi.org/10.3899/jrheum.121131DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3885333PMC
July 2013

The DBA/1 strain is a novel mouse model for experimental Borrelia burgdorferi infection.

Clin Vaccine Immunol 2012 Oct 1;19(10):1567-73. Epub 2012 Aug 1.

Department of Pediatrics, University of Pittsburgh School of Medicine, Pennsylvania, USA.

Lyme arthritis, caused by Borrelia burgdorferi, has similarities to rheumatoid arthritis and its experimental murine model, collagen-induced arthritis (CIA). Currently, no common strain exists for examination of arthritis models of Lyme arthritis and CIA, which are typically studied in C3H/HeJ and DBA/1 mice, respectively. The aim of this study was to define the characteristics of Borrelia burgdorferi infection and arthritis in the DBA/1 murine strain. Murine Lyme arthritis was induced in C3H/HeJ and DBA/1 mice by subcutaneous infection with B. burgdorferi. Tibiotarsal joints were measured during infection, and mice were sacrificed for histologic, microbiologic, and serologic analysis on days 14 and 42 postinfection. All bladder cultures obtained from C3H/HeJ and DBA/1 mice at 14 days postinfection grew Borrelia. There was no significant difference in spirochetal burdens in hearts and tibiotarsal joints at days 14 and 42 postinfection. Tibiotarsal joint swelling and histologic scoring were not significantly different between the two strains. Serologic analysis revealed increased IgG2a production in C3H/HeJ mice compared to DBA/1 mice. Analysis of 2-dimensional immunoblots revealed several specific antigens (LA7, BBA03, BBA64, BBA73, OspA, and VlsE) which were not recognized by DBA/1 sera. We conclude that the DBA/1 murine strain is a suitable model for the study of Lyme arthritis and experimental B. burgdorferi infection, allowing direct comparison between Lyme arthritis and collagen-induced arthritis. The specificity of the humoral immune response differs between the two strains, further study of which may reveal important findings about how individual strains respond to B. burgdorferi infection.
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http://dx.doi.org/10.1128/CVI.00251-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3485876PMC
October 2012

Do patients with juvenile idiopathic arthritis in clinical remission have evidence of persistent inflammation on 3T magnetic resonance imaging?

Arthritis Care Res (Hoboken) 2012 Dec;64(12):1846-54

Louisiana State University Health Science Center, New Orleans, USA.

Objective: Up to 90% of adults with rheumatoid arthritis (RA) in clinical remission have persistent synovitis and/or bone marrow lesions (BMLs) on magnetic resonance imaging (MRI). MRI findings in patients with juvenile idiopathic arthritis (JIA) in clinical remission have not been described. We utilized 3T MRI with contrast enhancement to examine JIA patients with hand and/or wrist involvement who were in clinical remission and compared them with a cohort of adult RA patients.

Methods: In total, 11 JIA patients and 10 RA patients with arthritis involving the hands and/or wrists were identified by their primary rheumatologist as being in physician-defined clinical remission, having no signs or symptoms of active arthritis and no medication changes for at least 6 months. A study rheumatologist performed a joint evaluation for tenderness, swelling, and limitation of motion, and study participants self-reported tender joint counts. The participants underwent MRI with intravenous contrast enhancement of 1 hand and wrist with a history of prior symptoms. A pediatric musculoskeletal radiologist blinded to the clinical data scored the MRIs for synovitis, tenosynovitis, and/or BMLs.

Results: Sixty-three percent of the JIA cohort and 70% of the RA cohort had MRI findings of synovitis, BMLs, and/or tenosynovitis. All pediatric patients with MRI abnormalities had normal physician tender and swollen joint counts. The patients' self-report of painful joint counts did not predict MRI abnormalities.

Conclusion: Over one-half of the patients in clinical remission had MRI evidence of persistent inflammation, defined as the presence of synovitis, tenosynovitis, or BMLs. A substantial portion of patients with JIA may have subclinical disease despite clinical remission.
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http://dx.doi.org/10.1002/acr.21774DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4398346PMC
December 2012

Plasma follistatin-like protein 1 is elevated in Kawasaki disease and may predict coronary artery aneurysm formation.

J Pediatr 2012 Jul 7;161(1):116-9. Epub 2012 Feb 7.

Division of Rheumatology, Children's Hospital of Pittsburgh, University of Pittsburgh, School of Medicine, Pittsburgh, PA.

Objective: To determine whether plasma levels of follistatin-like protein 1 (FSTL-1), a pro-inflammatory protein produced by mesenchymal tissue, including cardiac myocytes, correlate with the development of Kawasaki disease (KD) and coronary artery aneurysms (CAA).

Study Design: FSTL-1 plasma levels were measured serially with enzyme-linked immunosorbent assay in 48 patients with KD at time of diagnosis and, when available, 2 weeks, 6 weeks, and 6 months after onset of disease. These were compared with FSTL-1 plasma levels in 23 control subjects. Data were analyzed with generalized estimating equations.

Results: Plasma FSTL-1 levels were elevated in patients with acute KD compared with control subjects (P = .0086). FSTL-1 levels remained significantly elevated at 2 weeks after disease onset, but returned to control levels by 6 months. Seven patients with CAA had significantly higher FSTL-1 levels at the time of diagnosis than patients in whom aneurysms did not develop (P = .0018). Sensitivity and specificity rates for CAA at a specific FSTL-1 cutoff point (178 ng/mL) were 85% and 71%.

Conclusions: Plasma levels of FSTL-1 are elevated in acute KD and may predict cardiac morbidity in this disease. These results suggest a possible role for FSTL-1 in the formation of CAAs.
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http://dx.doi.org/10.1016/j.jpeds.2012.01.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3349793PMC
July 2012

FSTL1 promotes arthritis in mice by enhancing inflammatory cytokine/chemokine expression.

Arthritis Rheum 2012 Apr 17;64(4):1082-8. Epub 2011 Oct 17.

Children's Hospital of Pittsburgh, University of Pittsburgh Medical Center, University of Pittsburgh, Pittsburgh, Pennsylvania 15224, USA.

Objective: FSTL1 is a secreted glycoprotein that exacerbates murine arthritis and is overexpressed in human arthritis. The aim of this study was to determine the mechanism by which FSTL1 promotes arthritis.

Methods: Collagen-induced arthritis was induced in mice hypomorphic for FSTL1, generated with a gene trap-targeted mutant embryonic stem cell line. Arthritis was assessed by measuring paw swelling and using a qualitative arthritis index. Bone marrow-derived mesenchymal stromal cells from hypomorphic mice, as well as mouse stromal ST2 cells transduced with short hairpin RNA to suppress FSTL1 expression, were stimulated with interleukin-1β (IL-1β), tumor necrosis factor α, and IL-17. The monocyte cell line U937, which does not express FSTL1, was transfected with a plasmid encoding FSTL1 and stimulated with phorbol myristate acetate and lipopolysaccharide. Cell supernatants were assayed for IL-6, IL-8, monocyte chemotactic protein 1 (MCP-1), and FSTL1 by enzyme-linked immunosorbent assay.

Results: FSTL1 hypomorphic mice had reduced levels of FSTL1 compared to littermate controls. Following induction of arthritis, a significant correlation was observed between serum FSTL1 levels and both paw swelling and the arthritis index. Similar correlations were observed between the amount of FSTL1 produced by mesenchymal stromal cells, stromal ST2 cells, and monocytes and the secretion of IL-6, IL-8, and MCP-1.

Conclusion: These findings demonstrate that FSTL1 up-regulates proinflammatory mediators important in the pathology of arthritis, and that serum levels of FSTL1 correlate with severity of arthritis. The latter supports the possibility that FSTL1 might be a target for treatment of certain forms of arthritis.
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http://dx.doi.org/10.1002/art.33422DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3276726PMC
April 2012

Akt fine-tunes NF-κB-dependent gene expression during T cell activation.

J Biol Chem 2011 Oct 23;286(41):36076-36085. Epub 2011 Aug 23.

Dept. of Immunology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261. Electronic address:

Activation of the NF-κB signaling pathway is critical for leukocyte activation and development. Although previous studies suggested a role for the Akt kinase in coupling the T cell antigen receptor and CD28 to NF-κB activation in T cells, the nature of the role of Akt in this pathway is still unclear. Using a targeted gene profiling approach, we found that a subset of NF-κB-dependent genes required Akt for optimal up-regulation during T cell activation. The selective effects of Akt were manifest at the level of mRNA transcription and p65/RelA binding to upstream promoters and appear to be due to altered formation of the Carma1-Bcl10 complex. The proinflammatory cytokine TNF-α was found to be particularly sensitive to Akt inhibition or knockdown, including in primary human blood T cells and a murine model of rheumatoid arthritis. Our findings are consistent with a hierarchy in the expression of NF-κB-dependent genes, controlled by the strength and/or duration of NF-κB signaling. More broadly, our results suggest that defining the more graded effects of signaling, such as those demonstrated here for Akt and the NF-κB pathway, is important to understanding how cells can fine-tune signaling responses for optimal sensitivity and specificity.
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http://dx.doi.org/10.1074/jbc.M111.259549DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3195567PMC
October 2011

Follistatin-like protein 1 is a mesenchyme-derived inflammatory protein and may represent a biomarker for systemic-onset juvenile rheumatoid arthritis.

Arthritis Rheum 2010 Aug;62(8):2510-6

Children's Hospital of Pittsburgh of University of Pittsburgh Medical Center and University of Pittsburgh, Pittsburgh, Pennsylvania 15224, USA.

Objective: To examine both the source of follistatin-like protein 1 (FSTL-1) and the factors that induce its expression in arthritis, and to determine whether juvenile rheumatoid arthritis (JRA) is characterized by overexpression of FSTL-1.

Methods: FSTL-1 expression patterns were analyzed by immunohistochemical staining of joint tissue derived from mice with collagen-induced arthritis. Induction of FSTL-1 secretion was assessed in osteoblasts, adipocytes, and human fibroblast-like synoviocytes in response to transforming growth factor beta (TGFbeta), interleukin-1beta (IL-1beta), tumor necrosis factor alpha (TNFalpha), and IL-6. In addition, sera and synovial fluid from children with oligoarticular, polyarticular, or systemic-onset JRA were assayed for FSTL-1 using a custom enzyme-linked immunosorbent assay. FSTL-1 concentrations in these patients were assessed for correlations with the erythrocyte sedimentation rate (ESR) and platelet count.

Results: Immunohistochemical staining of murine joint sections demonstrated expression of FSTL-1 in all cell types of the mesenchymal lineage, including osteocytes, chondrocytes, adipocytes, and fibroblasts. FSTL-1 could be induced in osteoblasts, adipocytes, and human fibroblast-like synoviocytes by TGFbeta, IL-1beta, TNFalpha, and IL-6. The IL-1beta response was significantly greater than the TNFalpha response (P < 0.05). In human serum and synovial fluid, only those samples from children with the systemic-onset JRA subtype had elevated concentrations of FSTL-1. The synovial fluid concentrations of FSTL-1 were 2-3-fold higher than the serum concentrations. The elevation in serum FSTL-1 concentrations seen in children with systemic-onset JRA correlated closely with elevations in the ESR and platelet count.

Conclusion: These findings demonstrate that the arthritic joint matrix is a major source of FSTL-1 and that IL-1beta is a central mediator of FSTL-1 secretion. Furthermore, FSTL-1 may represent a useful biomarker of disease activity in systemic-onset JRA.
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http://dx.doi.org/10.1002/art.27485DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2921021PMC
August 2010

Synovial fluid proteins differentiate between the subtypes of juvenile idiopathic arthritis.

Arthritis Rheum 2010 Jun;62(6):1813-23

Children's Hospital of Pittsburgh, Division of Rheumatology, Pittsburgh, Pennsylvania 15213, USA.

Objective: Juvenile idiopathic arthritis (JIA) is a heterogeneous group of inflammatory diseases, and no clinically useful prognostic markers to predict disease outcome in children with JIA are currently available. Synovial fluid likely reflects the proteins present in the inflamed synovium. The purpose of this study was to delineate the synovial fluid proteome and determine whether protein expression differs in the different subtypes of JIA.

Methods: Synovial fluid samples obtained from children with oligoarticular JIA, polyarticular JIA, or systemic JIA were compared. Two-dimensional gel electrophoresis for protein separation and matrix-assisted laser desorption ionization-time-of-flight mass spectrometry and quadripole time-of-flight mass spectrometry for protein identification were used for this study. Synovial fluid cells were analyzed by polymerase chain reaction (PCR) for the presence of haptoglobin messenger RNA (mRNA).

Results: The synovial fluid proteome of the samples was delineated. The majority of proteins showed overexpression in JIA synovial fluid as compared with noninflammatory control samples. There were 24 statistically significantly differentially expressed spots (>2-fold change; P < 0.05) between the subtypes of JIA. PCR analysis revealed haptoglobin mRNA, suggesting that haptoglobin is locally produced in an inflamed joint in JIA.

Conclusion: Despite the similar histologic appearance of inflamed joints in patients with different subtypes of JIA, there are differences in protein expression according to the subtype of JIA. Haptoglobin is differentially expressed between the subtypes of JIA and is locally produced in an inflamed joint in JIA. Haptoglobin and other differentially expressed proteins may be potential biomarkers in JIA.
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http://dx.doi.org/10.1002/art.27447DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2918379PMC
June 2010

Pediatric pigmented villonodular synovitis mimicking a septic hip.

J Clin Rheumatol 2010 Mar;16(2):71-3

Division of Rheumatology, School of Medicine, Children's Hospital of Pittsburgh, University of Pittsburgh, Pittsburgh, PA 15201, USA.

We describe a child with pigmented villonodular synovitis initially treated for a presumed hip infection. The correct diagnosis was not made until 2(1/2) years later on a second admission. This is a rare disease with vague presenting symptoms that requires a high index of suspicion. Magnetic resonance imaging and tissue biopsy are usually needed for a definitive diagnosis. Surgery is the primary treatment option; however, the patient described was unusual in that she did well to date with conservative measures.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3947543PMC
http://dx.doi.org/10.1097/RHU.0b013e3181cf8657DOI Listing
March 2010

Loeys-Dietz syndrome in pregnancy: a case description and report of a novel mutation.

Fetal Diagn Ther 2009 10;26(1):35-7. Epub 2009 Oct 10.

Department of Obstetrics and Gynecology, Lis Maternity Hospital, Tel Aviv Sourasky Medical Center, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel.

Loeys-Dietz syndrome is a syndrome caused by heterozygous mutations in the genes encoding type 1 or 2 transforming growth factor-beta receptor (TGF-beta-R1/2). The obstetrical manifestations are risk of rupture of the gravid uterus and the arteries, either during pregnancy or in the immediate postpartum period, and damage to the vagina, perineum and the colon. We describe, for the first time, a new TGF-beta-R2 gene mutation in a family with several individuals who either had aortic rupture and dissection, sudden death or aortic root dilatation. The pregnancy was followed up and the baby was successfully delivered by a cesarean section at 34 weeks of gestation. The mother's recovery was uneventful and the baby was negative for the mutation on postnatal molecular testing. With appropriate supervision and early delivery by cesarean section, women with Loeys-Dietz syndrome can tolerate pregnancy and delivery without any adverse effect.
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http://dx.doi.org/10.1159/000236357DOI Listing
December 2009

Follistatin-like protein 1 promotes arthritis by up-regulating IFN-gamma.

J Immunol 2009 Jan;182(1):234-9

Division of Rheumatology, Children's Hospital of Pittsburgh, University of Pittsburgh, School of Medicine, Pittsburgh, PA 15213, USA.

Follistatin-like protein-1 (FSTL-1) is a poorly characterized protein that is up-regulated in the early stage of collagen-induced arthritis and that exacerbates arthritis when delivered by gene transfer. The current study was designed to determine the mechanism by which FSTL-1 promotes arthritis. FSTL-1 was injected into mouse paws, resulting in severe paw swelling associated with up-regulation of IFN-gamma transcript and the IFN-gamma-induced chemokine, CXCL10. Mice depleted of T cells were protected. A central role for IFN-gamma was confirmed by the finding that mice deficient in IFN-gamma failed to exhibit paw swelling in response to injection of FSTL-1. Furthermore, IFN-gamma secretion from mouse spleen cells exposed to a weak TCR signal was increased 5-fold in the presence of FSTL-1. FSTL-1 could be induced by innate immune signals, including TLR4 agonists and the arthritogenic cytokine, IL-1beta, via an NFkappaB pathway. Finally, FSTL-1 was found to be overexpressed in human arthritis and its neutralization inhibited murine collagen-induced arthritis and suppressed IFN-gamma and CXCL10 production in arthritic joints. These findings demonstrate that FSTL-1 plays a critical role in arthritis by enhancing IFN-gamma signaling pathways and suggest a mechanism by which FSTL-1 bridges innate and adaptive immune responses.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3150499PMC
http://dx.doi.org/10.4049/jimmunol.182.1.234DOI Listing
January 2009

Is long-term etanercept therapy safe and effective in patients with juvenile RA?

Authors:
Raphael Hirsch

Nat Clin Pract Rheumatol 2008 Dec 14;4(12):628-9. Epub 2008 Oct 14.

Pediatric Rheumatology, Children's Hospital of Pittsburgh , University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA.

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http://dx.doi.org/10.1038/ncprheum0925DOI Listing
December 2008

Three-dimensional and thermal surface imaging produces reliable measures of joint shape and temperature: a potential tool for quantifying arthritis.

Arthritis Res Ther 2008 23;10(1):R10. Epub 2008 Jan 23.

Division of Rheumatology, Children's Hospital of Pittsburgh, 3705 Fifth Avenue, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA.

Introduction: The assessment of joints with active arthritis is a core component of widely used outcome measures. However, substantial variability exists within and across examiners in assessment of these active joint counts. Swelling and temperature changes, two qualities estimated during active joint counts, are amenable to quantification using noncontact digital imaging technologies. We sought to explore the ability of three dimensional (3D) and thermal imaging to reliably measure joint shape and temperature.

Methods: A Minolta 910 Vivid non-contact 3D laser scanner and a Meditherm med2000 Pro Infrared camera were used to create digital representations of wrist and metacarpalphalangeal (MCP) joints. Specialized software generated 3 quantitative measures for each joint region: 1) Volume; 2) Surface Distribution Index (SDI), a marker of joint shape representing the standard deviation of vertical distances from points on the skin surface to a fixed reference plane; 3) Heat Distribution Index (HDI), representing the standard error of temperatures. Seven wrists and 6 MCP regions from 5 subjects with arthritis were used to develop and validate 3D image acquisition and processing techniques. HDI values from 18 wrist and 9 MCP regions were obtained from 17 patients with active arthritis and compared to data from 10 wrist and MCP regions from 5 controls. Standard deviation (SD), coefficient of variation (CV), and intraclass correlation coefficients (ICC) were calculated for each quantitative measure to establish their reliability. CVs for volume and SDI were <1.3% and ICCs were greater than 0.99.

Results: Thermal measures were less reliable than 3D measures. However, significant differences were observed between control and arthritis HDI values. Two case studies of arthritic joints demonstrated quantifiable changes in swelling and temperature corresponding with changes in symptoms and physical exam findings.

Conclusion: 3D and thermal imaging provide reliable measures of joint volume, shape, and thermal patterns. Further refinement may lead to the use of these technologies to improve the assessment of disease activity in arthritis.
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http://dx.doi.org/10.1186/ar2360DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2374475PMC
July 2008

Gene therapy for arthritis.

Mod Rheumatol 2008 5;18(1):2-14. Epub 2008 Jan 5.

Division of Rheumatology, Children's Hospital of Pittsburgh, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA.

Arthritis is among the leading causes of disability in the developed world. There remains no cure for this disease and the current treatments are only modestly effective at slowing the disease's progression and providing symptomatic relief. The clinical effectiveness of current treatment regimens has been limited by short half-lives of the drugs and the requirement for repeated systemic administration. Utilizing gene transfer approaches for the treatment of arthritis may overcome some of the obstacles associated with current treatment strategies. The present review examines recent developments in gene therapy for arthritis. Delivery strategies, gene transfer vectors, candidate genes, and safety are also discussed.
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http://dx.doi.org/10.1007/s10165-007-0017-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2275302PMC
May 2008

Staphylococcus aureus panniculitis complicating juvenile dermatomyositis.

Pediatrics 2007 Feb 29;119(2):e528-30. Epub 2007 Jan 29.

Division of Rheumatology, Children's Hospital of Pittsburgh, 3705 Fifth Ave, Pittsburgh, PA 15213, USA.

Panniculitis is a rarely reported manifestation of juvenile dermatomyositis. The 3 previously reported cases of juvenile dermatomyositis and panniculitis were attributed to flare of underlying disease, rather than infection, and were treated with increased immunosuppression. Here we describe a patient with juvenile dermatomyositis who developed panniculitis secondary to Staphylococcus aureus. Patients with juvenile dermatomyositis and panniculitis should have extensive testing for infectious etiologies before increasing their immunosuppressive regimens.
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http://dx.doi.org/10.1542/peds.2006-1518DOI Listing
February 2007

Recombinant adeno-associated virus preferentially transduces human, compared to mouse, synovium: implications for arthritis therapy.

Mod Rheumatol 2004 ;14(1):18-24

William S. Rowe Division of Rheumatology, Children’s Hospital Medical Center, Cincinnati, OH, USA.

Despite a number of published reports, including from our own laboratory, suggesting that adeno-associated virus (AAV) transduces mouse synovium, a careful analysis demonstrated transduction predominantly of the subsynovial muscle tissue, while the synovial lining is poorly transduced. To investigate the potential of AAV to transduce human synovium, three human rheumatoid arthritis (RA) and two murine collagen-induced arthritis (CIA) synovial cell lines were infected with recombinant AAV (rAAV) vectors encoding either mouse IL-10 or IL-4. Low-level transgene expression was observed. However, either Gamma-irradiation or the addition of a low-titer E1-, E3-deleted recombinant adenovirus resulted in up to a 100-fold increase in transgene product in the human, but not the mouse, cell lines. RA synovial tissues implanted subcutaneously in severe combined immunodeficiency (SCID) mice, which were subsequently infected with rAAV, showed marked increases in transgene expression when co-infected with adenovirus. To our knowledge, this is the first study to show that intact human synovial tissues can be transduced by rAAV, and it suggests that murine arthritis may not be an optimal model to study rAAV as a gene transfer vector. Further studies to elucidate the mechanisms limiting gene transduction in human synovium may allow optimization of this vector for the treatment of arthritis.
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http://dx.doi.org/10.1007/s10165-003-0260-7DOI Listing
October 2012

Follistatin-like protein-1 is a novel proinflammatory molecule.

J Immunol 2006 Oct;177(7):4758-62

Division of Rheumatology, Children's Hospital of Pittsburgh, School of Medicine, University of Pittsburgh, 3705 Fifth Avenue, Pittsburgh, PA 15213, USA.

While analyzing gene expression in collagen-induced arthritis, we discovered that a poorly characterized gene, follistatin-like protein 1 (FSTL-1), is highly overexpressed in mouse paws during early arthritis, especially at the interface of synovial pannus and eroding bone. In this study, we show that FSTL-1 is a novel proinflammatory molecule with a previously unrecognized role in inflammation. Transfection of FSTL-1 into macrophages and fibroblasts leads to up-regulation of proinflammatory cytokines, including IL-1beta, TNF-alpha, and IL-6. Overexpression of FSTL-1 in mouse paws by gene transfer results in severe paw swelling and arthritis.
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http://dx.doi.org/10.4049/jimmunol.177.7.4758DOI Listing
October 2006

Inflammatory cytokine regulation of transgene expression in human fibroblast-like synoviocytes infected with adeno-associated virus.

Arthritis Rheum 2006 Jul;54(7):2119-26

University of Pittsburgh, Pittsburgh, Pennsylvania, USA.

Objective: An ideal gene transfer vector for chronic inflammatory diseases such as rheumatoid arthritis (RA) would provide local transgene expression only when the disease is active. To determine whether adeno-associated virus (AAV) possesses this ability, the effects of inflammatory cytokines on transgene expression were evaluated in human RA fibroblast-like synoviocytes (FLS).

Methods: Human FLS were infected with AAV in the presence or absence of inflammatory cytokines or synovial fluid obtained from patients with RA. Transgene expression was monitored by either enzyme-linked immunosorbent assay or flow cytometry. Transgene messenger RNA (mRNA) was measured by real-time quantitative reverse transcription-polymerase chain reaction.

Results: Inflammatory cytokines increased transgene expression in FLS by up to 60-fold. Synovial fluid from patients with RA, but not from patients without arthritis, was also able to increase expression in synoviocytes. Protein expression correlated with transgene mRNA levels. The enhanced expression required the continued presence of cytokines because, upon removal, transgene expression returned to baseline levels. Expression could be repeatedly reinduced by reexposure to cytokines. The effect was not promoter specific and was demonstrated to be phosphatidylinositol 3-kinase-dependent.

Conclusion: These results suggest that expression of a therapeutic transgene can be controlled by the presence of inflammation following AAV gene transfer, making it an attractive vector for chronic inflammatory diseases such as RA.
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http://dx.doi.org/10.1002/art.21940DOI Listing
July 2006

NOD.c3c4 congenic mice develop autoimmune biliary disease that serologically and pathogenetically models human primary biliary cirrhosis.

J Exp Med 2006 May 24;203(5):1209-19. Epub 2006 Apr 24.

Division of Rheumatology and Immunology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA.

Primary biliary cirrhosis (PBC) is an autoimmune disease with a strong genetic component characterized by biliary ductular inflammation with eventual liver cirrhosis. The serologic hallmark of PBC is antimitochondrial antibodies that react with the pyruvate dehydrogenase complex, targeting the inner lipoyl domain of the E2 subunit (anti-PDC-E2). Herein we demonstrate that NOD.c3c4 mice congenically derived from the nonobese diabetic strain develop an autoimmune biliary disease (ABD) that models human PBC. NOD.c3c4 (at 9-10 wk, before significant biliary pathology) develop antibodies to PDC-E2 that are specific for the inner lipoyl domain. Affected areas of biliary epithelium are infiltrated with CD3+, CD4+, and CD8+ T cells, and treatment of NOD.c3c4 mice with monoclonal antibody to CD3 protects from ABD. Furthermore, NOD.c3c4-scid mice develop disease after adoptive transfer of splenocytes or CD4+ T cells, demonstrating a central role for T cells in pathogenesis. Histological analysis reveals destructive cholangitis, granuloma formation, and eosinophilic infiltration as seen in PBC, although, unlike PBC, the extrahepatic biliary ducts are also affected. Using a congenic mapping approach, we define the first ABD (Abd) locus, Abd1. These results identify the NOD.c3c4 mouse as the first spontaneous mouse model of PBC.
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http://dx.doi.org/10.1084/jem.20051911DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2121204PMC
May 2006