Viability and apoptosis of human chondrocytes in osteochondral fragments following joint trauma.

Post-traumatic arthritis is a frequent consequence of articular fracture. The mechanisms leading to its development after such injuries have not been clearly delineated. A potential contributing factor is decreased viability of the articular chondrocytes. The object of this study was to characterise the regional variation in the viability of chondrocytes following joint trauma. A total of 29 osteochondral fragments from traumatic injuries to joints that could not be used in articular reconstruction were analysed for cell viability using the fluorescence live/dead assay and for apoptosis employing the TUNEL assay, and compared with cadaver control fragments. Chondrocyte death and apoptosis were significantly greater along the edge of the fracture and in the superficial zone of the osteochondral fragments. The middle and deep zones demonstrated significantly higher viability of the chondrocytes. These findings indicate the presence of both necrotic and apoptotic chondrocytes after joint injury and may provide further insight into the role of chondrocyte death in post-traumatic arthritis.

Heterodimerization between the glucocorticoid receptor and the unrelated DNA-binding protein, Xenopus glucocorticoid receptor accessory factor.

The adrenal steroid hormones, glucocorticoids, control many physiological responses to trauma, including elevated synthesis of fibrinogen, a major blood-clotting protein. Glucocorticoid regulation of the gamma-fibrinogen subunit gene in Xenopus laevis is mediated by a binding site for Xenopus glucocorticoid receptor accessory factor (XGRAF) and a contiguous glucocorticoid response element (GRE) half-site. Here, we characterize the protein:DNA complex formed by a cooperative interaction between XGRAF, GR, and the DNA. We demonstrate that the complex contains XGRAF by competition in a gel shift assay. The presence of GR is established by two criteria: 1) size dependence of the XGRAF:GR:DNA complex on the size of the GR component and 2) interference with complex formation by GR antibody. Cooperative binding of XGRAF and GR to the DNA was quantitated, showing that GR favors binding to XGRAF:DNA compared with free DNA by a factor of 30. The cooperative interaction between XGRAF and GR can occur on nicked DNA but is disrupted when 1 bp is inserted between the XGRAF binding site and half-GRE. Significantly, this loss of physical association in vitro correlates with loss of XGRAF amplification of GR activity in transiently transfected primary Xenopus hepatocytes. The simplest explanation for cooperativity between XGRAF and GR is formation of a DNA-bound heterodimer of these two proteins. This mechanism represents a new mode of transcriptional regulation in which GR and a nonreceptor protein form a heterodimer, with both partners contacting their specific DNA sites simultaneously.

The binding site for Xenopus glucocorticoid receptor accessory factor and a single adjacent half-GRE form an independent glucocorticoid response unit.

In Xenopus laevis, transcription of the gamma-fibrinogen subunit gene is activated by glucocorticoids. Hormone induction is regulated by three glucocorticoid response element (GRE) half-sites and an additional DNA sequence which binds a novel hepatocyte nuclear protein, Xenopus glucocorticoid receptor accessory factor (XGRAF). The XGRAF binding site (GAGTTAA) is located directly upstream of the most distal half-GRE. The proximity of the binding sites for XGRAF and the glucocorticoid receptor (GR) led to the hypothesis that these two sites form a glucocorticoid response unit (GRU). By transfecting DNA into primary hepatocytes, we showed that this GRU confers hormone responsiveness in the absence of other half-GREs. The XGRAF binding site enhances function of the half-GRE without itself responding to glucocorticoids. The GRU retains efficacy in other locations relative to the gamma-fibrinogen gene promoter, further increases transcription when present in multiple copies, and activates a heterologous promoter. Despite the contiguity of the XGRAF binding site and half-GRE, the two sites can be occupied simultaneously in vitro. The binding characteristics correlate with function since mutations that disrupt concurrent XGRAF and GR binding also impair transcription. This novel GRU represents a new regulatory mechanism that may be applicable to other glucocorticoid responsive genes that lack a full GRE.

Analysis of the DNA-binding site for Xenopus glucocorticoid receptor accessory factor. Critical nucleotides for binding specificity in vitro and for amplification of steroid-induced fibrinogen gene transcription.

In addition to the glucocorticoid receptor, DNA-binding proteins called accessory factors play a role in hormone activation of many glucocorticoid-responsive genes. Hormonal regulation of the gamma-fibrinogen subunit gene from the frog Xenopus laevis requires a novel DNA sequence that binds a liver nuclear protein called Xenopus glucocorticoid receptor accessory factor (XGRAF). Here we demonstrate that the recognition site for XGRAF encompasses GAGTTAA at positions -175 to -169 relative to the start site of transcription. This sequence is not closely related to the binding sites for known transcription factors. The two guanosines make close contact with XGRAF, as shown by the methylation interference assay. Single-point mutagenesis of every nucleotide in the 9-base pair region from positions -177 to -169 showed an excellent correlation between ability to bind XGRAF in vitro and ability to amplify hormone-induced transcription from DNA transfected into Xenopus primary hepatocytes. Conversely, XGRAF had little or no effect on basal transcription of the gamma-fibrinogen gene. Maximal hormonal induction also requires three half-glucocorticoid response elements (half-GREs) homologous to the downstream half of the consensus GRE. Interestingly, the XGRAF-binding site is immediately adjacent to the most important half-GRE. This close proximity suggests a new mechanism for activation of a gene lacking a conventional full GRE.

A phase I/II open label study of the safety and efficacy of an anti-ICAM-1 (intercellular adhesion molecule-1; CD54) monoclonal antibody in early rheumatoid arthritis.

OBJECTIVE: Previous work suggested the potential utility of therapy with a monoclonal antibody (Mab) to intercellular adhesion molecule-1 (ICAM-1; CD54) in patients with longstanding rheumatoid arthritis (RA). Immunomodulatory interventions, including adhesion receptor directed therapies, might be expected to have greater efficacy in patients with less established or less aggressive disease. Therefore, we assessed the efficacy and safety of an anti-ICAM-1 Mab in patients with early RA. METHODS: An open label study of a 5 day infusion of an anti-ICAM-1 Mab in 10 patients with early or indolent RA was conducted. These patients were defined as having previously used < or = 1 disease modifying antirheumatic drug. RESULTS: Based on composite criteria, 7/10 patients had a marked or moderate response to therapy at one month of followup. Clinical benefit was sustained through 2 months for 5/10 patients and 3/10 had extended benefit (11, 8, and > 7 months). Clinical benefit was more likely to be obtained in patients with subacute onset of disease than in those with a fulminant onset. CONCLUSION: A single course of therapy with an anti-ICAM-1 Mab was associated with clinical improvement in a group of patients with early or indolent RA to an extent apparently greater than previously observed in patients with longstanding, aggressive RA.

cDNA and amino-acid sequences and organization of the gene encoding the B beta subunit of fibrinogen from Xenopus laevis.

Fibrinogen, the major blood-clotting protein, is made up of three chains, A alpha, B beta and gamma, which are synthesized and secreted by the liver. In this communication, we describe the complete cDNA sequence, deduced amino acid (aa) sequence and organization of the gene encoding the B beta subunit of fibrinogen from Xenopus laevis (Xl). The cDNA representing the predominant form of the B beta mRNA comprises 2390 nucleotides (nt), with an open reading frame of 1467 nt coding for a 488-aa protein. The percent identity between Xl B beta and that of other animals ranges from 50% for lamprey to 66% for human. The Xl B beta gene consists of nine exons, one more than found in the human gene. The exon/intron boundaries in the frog and human B beta genes are in exactly conserved positions, except for junctions in the highly variable fibrinopeptide-encoding regions. Three of the exon/intron boundaries in the Xl B beta gene are also analogous to ones in A alpha and gamma genes of other species, supporting the notion of a close evolutionary relationship between the genes for all three subunits. This analysis of B beta from an amphibian provides the first complete description of the arrangement of exons and introns in any fibrinogen subunit gene from a non mammal and gives insight into the most highly conserved aspects of fibrinogen protein structure and gene organization.

Induction of persistent T cell hyporesponsiveness in vivo by monoclonal antibody to ICAM-1 in patients with rheumatoid arthritis.

Rheumatoid arthritis patients undergoing treatment with a murine mAb (BIRR1) to ICAM-1 were studied to determine the effects of the treatment on T cell responsiveness assayed in vitro. Previous studies had demonstrated that over the 5-day treatment period, there was a transient increase in circulating T cells that returned to base line 3 days after therapy. The transient lymphocytosis correlated with a loss in delayed-type hypersensitivity reactivity during the time of Ab administration. However, neither the increase in T cell numbers nor the inhibition of delayed-type hypersensitivity responses correlated with the immediate clinical benefit of therapy or the prolonged nature of the response to therapy in some patients. The current studies show that after the return of lymphocyte numbers to pretreatment levels, a decrease in T lymphocyte responses to suboptimal activation signals, including accessory cell-dependent (low dose PHA and soluble anti-CD3 mAb) and accessory cell-independent stimuli (immobilized anti-CD3 mAb), was observed. However, responses to recall Ags were preserved. Depressed T cell responses were not the result of diminished accessory cell function or production of suppressive factors by monocytes, but rather reflected decreased IL-2 production. The duration of T cell hyporesponsiveness was variable in length but lasted up to 5 mo after treatment with anti-ICAM-1 mAb. The induction and persistence of T cell hyporesponsiveness correlated with an improvement in disease activity in treated patients. These studies show that treatment with anti-ICAM-1 mAb can induce T cell hyporesponsiveness that correlates with and may explain sustained therapeutic benefit in patients with rheumatoid arthritis.

Treatment of refractory rheumatoid arthritis with a monoclonal antibody to intercellular adhesion molecule 1.

OBJECTIVE: To assess the safety and efficacy of a monoclonal antibody (MAb) to intercellular adhesion molecule 1 (ICAM-1; CD54) in rheumatoid arthritis (RA). METHODS: A phase I/II, open-label, dose-escalation study of 32 patients. RESULTS: During treatment, a peripheral CD3+/CD4+ lymphocytosis was noted, and several patients demonstrated transient cutaneous anergy, which suggests that therapy modified T cell recirculation. Thirteen of the 23 patients who received 5 days of treatment demonstrated clinical improvement through day 29, and 9 of 23 through day 60. Adverse effects were minor and transient. CONCLUSION: Anti-ICAM-1 MAb therapy was well tolerated, resulted in a transient alteration in T lymphocyte recirculation, and effected clinical improvement in some RA patients.

Transcriptional regulation of the Xenopus laevis B beta fibrinogen subunit gene by glucocorticoids and hepatocyte nuclear factor 1: analysis by transfection into primary liver cells.

The blood-clotting protein fibrinogen is composed of three subunits, designated A alpha, B beta, and gamma, which are encoded by a family of related genes. As part of the acute-phase response, expression of the fibrinogen genes is coordinately regulated in the liver by glucocorticoids. To understand the factors underlying this hormonal response, we have examined control of transcription from fibrinogen gene fragments transfected into hepatocytes from the frog Xenopus laevis. This analysis is the first in any species to define transcriptional regulatory elements for the fibrinogen genes by transfection into primary liver cells, rather than liver-derived cell lines. A transfection vector was constructed containing the Xenopus B beta gene transcription start site and 1293 bp of the 5' flanking sequence linked to the firefly luciferase gene. When this construct was transfected into primary liver parenchymal cells, luciferase expression was induced approximately 14-fold by glucocorticoids, an increase similar to the transcriptional stimulation of the endogenous B beta subunit gene. DNA fragments with as little as 284 bases of upstream sequence retained full hormone responsiveness. This region contains a sequence resembling the canonical glucocorticoid response element (GRE) at bases -148 to -162. Deletions or specific point mutations eliminating this putative GRE led to complete loss of glucocorticoid inducibility. Physical association of the steroid hormone receptor with this functional GRE was demonstrated with a truncated form of the rat glucocorticoid receptor containing the DNA-binding domain. A second possible GRE at positions -526 to -540 was not hormone-responsive, in either the presence or the absence of the more proximal GRE. The regulatory region also has a sequence similar to the binding site for a liver-specific transcription factor, hepatocyte nuclear factor 1 (HNF-1), at positions -120 to -132. Specific point mutations in the HNF-1-binding site, in a construct containing a wild-type GRE, reduced promoter activity by a factor of 10, while stimulation by glucocorticoids was retained. Binding studies confirmed specific interaction between this site and the transcription factor HNF-1 alpha from mouse. Thus, we have identified a GRE sufficient to account for full glucocorticoid inducibility and an HNF-1 site close to the promoter that are major determinants of transcriptional control of the Xenopus fibrinogen B beta subunit gene in cells from normal liver tissue.