Homogentisate 1,2 Dioxygenase is Expressed in Human Osteoarticular Cells: Implications in Alkaptonuria – ScienceOpen
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      Homogentisate 1,2 Dioxygenase is Expressed in Human Osteoarticular Cells: Implications in Alkaptonuria

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          Abstract

          Alkaptonuria (AKU) results from defective homogentisate1,2-dioxygenase (HGD), causing degenerative arthropathy. The deposition of ochronotic pigment in joints is so far attributed to homogentisic acid produced by the liver, circulating in the blood and accumulating locally. Human normal and AKU osteoarticular cells were tested for HGD gene expression by RT-PCR, mono- and 2D-Western blotting. HGD gene expression was revealed in chondrocytes, synoviocytes, osteoblasts. Furthermore, HGD expression was confirmed by Western blotting, that also revealed the presence of five enzymatic molecular species. Our findings indicate that AKU osteoarticular cells produce the ochronotic pigment in loco and this may strongly contribute to induction of ochronotic arthropathy. J. Cell. Physiol. 227: 3254–3257, 2012. © 2011 Wiley Periodicals, Inc.

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          The American College of Rheumatology criteria for the classification and reporting of osteoarthritis of the hip.

          Clinical criteria for the classification of patients with hip pain associated with osteoarthritis (OA) were developed through a multicenter study. Data from 201 patients who had experienced hip pain for most days of the prior month were analyzed. The comparison group of patients had other causes of hip pain, such as rheumatoid arthritis or spondylarthropathy. Variables from the medical history, physical examination, laboratory tests, and radiographs were used to develop different sets of criteria to serve different investigative purposes. Multivariate methods included the traditional "number of criteria present" format and "classification tree" techniques. Clinical criteria: A classification tree was developed, without radiographs, for clinical and laboratory criteria or for clinical criteria alone. A patient was classified as having hip OA if pain was present in combination with either 1) hip internal rotation greater than or equal to 15 degrees, pain present on internal rotation of the hip, morning stiffness of the hip for less than or equal to 60 minutes, and age greater than 50 years, or 2) hip internal rotation less than 15 degrees and an erythrocyte sedimentation rate (ESR) less than or equal to 45 mm/hour; if no ESR was obtained, hip flexion less than or equal to 115 degrees was substituted (sensitivity 86%; specificity 75%). Clinical plus radiographic criteria: The traditional format combined pain with at least 2 of the following 3 criteria: osteophytes (femoral or acetabular), joint space narrowing (superior, axial, and/or medial), and ESR less than 20 mm/hour (sensitivity 89%; specificity 91%). The radiographic presence of osteophytes best separated OA patients and controls by the classification tree method (sensitivity 89%; specificity 91%). The "number of criteria present" format yielded criteria and levels of sensitivity and specificity similar to those of the classification tree for the combined clinical and radiographic criteria set. For the clinical criteria set, the classification tree provided much greater specificity. The value of the radiographic presence of an osteophyte in separating patients with OA of the hip from those with hip pain of other causes is emphasized.
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            Cytokines in chronic inflammatory arthritis. V. Mutual antagonism between interferon-gamma and tumor necrosis factor-alpha on HLA-DR expression, proliferation, collagenase production, and granulocyte macrophage colony-stimulating factor production by rheumatoid arthritis synoviocytes.

            The effects of a broad array of cytokines, individually and in combination, were determined on separate functions (proliferation, collagenase production, and granulocyte macrophage colony-stimulating factor [GM-CSF] production) and phenotype (expression of class II MHC antigens) of cultured fibroblast-like RA synoviocytes. The following recombinant cytokines were used: IL-1 beta, IL-2, IL-3, IL-4, IFN-gamma, tumor necrosis factor (TNF)-alpha, GM-CSF, and macrophage colony-stimulating factor (M-CSF). Only IFN-gamma induced HLA-DR (but not HLA-DQ) expression. TNF-alpha inhibited IFN-gamma-mediated HLA-DR expression (46.7 +/- 4.1% inhibition) and HLA-DR mRNA accumulation. This inhibitory effect was also observed in osteoarthritis synoviocytes. Only TNF-alpha and IL-1 increased synoviocyte proliferation (stimulation index 3.60 +/- 1.03 and 2.31 +/- 0.46, respectively). IFN-gamma (but none of the other cytokines) inhibited TNF-alpha-induced proliferation (70 +/- 14% inhibition) without affecting the activity of IL-1. Only IL-1 beta and TNF-alpha induced collagenase production (from less than 0.10 U/ml to 1.10 +/- 0.15 and 0.72 +/- 0.24, respectively). IFN-gamma decreased TNF-alpha-mediated collagenase production (69 +/- 19% inhibition) and GM-CSF production but had no effect on the action of IL-1. These data demonstrate mutual antagonism between IFN-gamma and TNF-alpha on fibroblast-like synoviocytes and suggest a novel homeostatic control mechanism that might be defective in RA where very little IFN-gamma is produced.
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              Development of an in vitro model to investigate joint ochronosis in alkaptonuria.

              Alkaptonuria (AKU) is a genetic disorder caused by lack of the enzyme responsible for breaking down homogentisic acid (HGA), an intermediate in tyrosine metabolism. HGA is deposited as a polymer, termed ochronotic pigment, in collagenous tissues. Pigmentation is progressive over many years, leading to CTDs including severe arthropathies. To investigate the mechanism of pigmentation and to determine how it leads to arthropathy, we aimed to develop an in vitro model of ochronosis. Osteosarcoma cell lines MG63, SaOS-2 and TE85 were cultured in medium containing HGA from 0.1 μM to 1 mM. Cultures were examined by light microscopy and transmission electron microscopy, and Schmorl's stain was used to detect pigment deposits in vitro, following the observation that this stain identifies ochronotic pigment in AKU tissues. The effects of HGA on cell growth and collagen synthesis were also determined. There was a dose-related deposition of pigment in cells and associated matrix from 33 μM to 0.33 mM HGA. Pigmentation in vitro was much more rapid than in vivo, indicating that protective mechanisms exist in tissues in situ. Pigment deposition was dependent on the presence of cells and was observed at HGA concentrations that were not toxic. There was an inhibition of cell growth and a stimulation of type I collagen synthesis up to 0.33 mM HGA, but severe cell toxicity at 1 mM HGA. We have developed an in vitro model of ochronosis that should contribute to understanding joint destruction in AKU and to the aetiology of OA.
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                Author and article information

                Journal
                J Cell Physiol
                J. Cell. Physiol
                jcp
                Journal of Cellular Physiology
                Wiley Subscription Services, Inc., A Wiley Company (Hoboken )
                0021-9541
                1097-4652
                September 2012
                21 November 2011
                : 227
                : 9
                : 3254-3257
                Affiliations
                [1 ]simpleDipartimento di Biotecnologie, Università degli Studi di Siena Siena Italy
                [2 ]simpleCentro Interdipartimentale per lo Studio Biochimico delle Patologie Osteoarticolari, Università degli Studi di Siena Siena Italy
                [3 ]simpleToscana Life Sciences Foundation Siena Italy
                Author notes
                *Correspondence to: Annalisa Santucci, Università degli Studi di Siena, Dipartimento di Biotecnologie, via Fiorentina 1, 53100 Siena (SI), Italy. E-mail: santucci@ 123456unisi.it

                Marcella Laschi and Laura Tinti equally contributed to this work.

                Contract grant sponsor: Telethon;

                Contract grant number: GGP10058.

                Contract grant sponsor: Toscana Life Sciences Foundation project Orphan;

                Contract grant number: 0108.

                Contract grant sponsor: Fondazione Monte dei Paschi di Siena;

                Contract grant number: Esercizio 2008-2009-2010.

                Article
                10.1002/jcp.24018
                3427883
                22105303
                90586902-3b4d-47c3-99e3-38a1dc9fbde8
                Copyright © 2011 Wiley Periodicals, Inc.

                Re-use of this article is permitted in accordance with the Creative Commons Deed, Attribution 2.5, which does not permit commercial exploitation.

                History
                : 26 October 2011
                : 15 November 2011
                Categories
                Original Research Articles

                Anatomy & Physiology
                Anatomy & Physiology

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