Efficacy of a collagen-based dressing in an animal model of delayed wound healing.

OBJECTIVE: The aim of this study was to evaluate in vitro and in vivo the efficacy of GBT013, a collagen-based dressing, for the treatment of chronic wounds, in a db/db mouse model of diabetes. METHOD: Macroscopic and histologic analyses of db/db mice wound healing with GBT013 or saline gauze were assessed. The mRNA expression and the proliferation of dermal fibroblast were investigated. Matrix metalloproteinases (MMP)-2 and MMP-9 activities were quantified. RESULTS: In db/db mice, GBT013 improves wound epithelialisation when compared with saline gauze. Histological analysis of scar tissue also shows an enhancement of remodelling associated with no sign of acute inflammation. In addition, GBT013 significantly decreases interleukin (IL)-6 and IL-8, significantly increases tissue inhibitors of metalloproteinases (TIMP)-1 and TIMP-2 fibroblast mRNA expression and significantly reduces in vitro MMP-2 and MMP-9 enzymatic activities. Moreover, GBT013 allows cell growth inside the matrix and stimulates proliferation of human dermal fibroblast. CONCLUSION: By contributing to restore MMPs/TIMPs balance, GBT013 may function in all key stages of wound healing, such as inflammation, proliferation and tissue remodelling, and ultimately may provide a favourable environment for skin repair. DECLARATION OF INTEREST: This work was supported by Genbiotech, the R&D subsidiary of Laboratoires Genévrier, a pharmaceutical company.

Increased pregnancy rate using standardized coculture on autologous endometrial cells and single blastocyst transfer : a multicentre randomized controlled trial.

Despite excellent published results, the lack of well-designed, multicentre, randomized clinical trials results in an absence of general consensus on the efficacy of autologous endometrial cells coculture (AECC) in Assisted Reproductive Technology (ART). An open, multicentre, prospective, randomized controlled trial was designed to compare the pregnancy rate (PR) after the transfer of one blastocyst on day 5 after AECC to the transfer of one embryo on day 3 (control group). Patients were women aged 18 to 36, undergoing an ART cycle with no more than 1 embryo transfer failure. Sample size was calculated at 720 for a superiority trial involving an intermediate analysis at 300 patients. We present the results of the intermediate analysis that resulted in the study ending considering the observed difference. Three hundred thirty nine patients were randomized: 170 in the AECC group and 169 in the control group. The clinical PR per transfer was 53.4% with AECC and 37.3% in the control group (p=0.025). The quality of embryos was improved with AECC. These results suggest that implementation of the AECC technique to a large number of In-Vitro Fertilization (IVF) centres could lead to a substantial improvement in the proportion of successful assisted reproduction. The study was supported by the Laboratoires Genévrier, France.

scAAV-mediated gene transfer of interleukin-1-receptor antagonist to synovium and articular cartilage in large mammalian joints.

With the long-term goal of developing a gene-based treatment for osteoarthritis (OA), we performed studies to evaluate the equine joint as a model for adeno-associated virus (AAV)-mediated gene transfer to large, weight-bearing human joints. A self-complementary AAV2 vector containing the coding regions for human interleukin-1-receptor antagonist (hIL-1Ra) or green fluorescent protein was packaged in AAV capsid serotypes 1, 2, 5, 8 and 9. Following infection of human and equine synovial fibroblasts in culture, we found that both were only receptive to transduction with AAV1, 2 and 5. For these serotypes, however, transgene expression from the equine cells was consistently at least 10-fold higher. Analyses of AAV surface receptor molecules and intracellular trafficking of vector genomes implicate enhanced viral uptake by the equine cells. Following delivery of 1 × 10(11) vector genomes of serotypes 2, 5 and 8 into the forelimb joints of the horse, all three enabled hIL-1Ra expression at biologically relevant levels and effectively transduced the same cell types, primarily synovial fibroblasts and, to a lesser degree, chondrocytes in articular cartilage. These results provide optimism that AAV vectors can be effectively adapted for gene delivery to large human joints affected by OA.

A simple, lanthanide-based method to enhance the transduction efficiency of adenovirus vectors.

Based upon the powerful bridging and charge-masking properties of lanthanide cations (Ln3+), we have investigated their use to improve the transduction efficiency of adenovirus vectors. Using a luciferase marker gene, it was possible to increase transgene expression by the murine mesenchymal stem cell line C3H10T(1/2) by up to four log orders when using very low multiplicities of infection in conjunction with Ln3+; La3+ was superior to Gd3+, Y3+ and Lu3+ in this regard. All Ln3+ were more effective than Ca2+. Flow cytometry, using a green fluorescent protein marker gene, confirmed that La3+ increased both the percentage of transduced cells and the level of transgene expression per cell. Transduction of primary cultures of a variety of different mesenchymal cells from human, rabbit, bovine and rat sources, as well as gene transfer to synovium and muscle in vivo, was also greatly enhanced. Our findings suggest that this lanthanide-based method holds much promise for expediting both experimental and clinical applications of gene transfer with adenoviral vectors.

cDNA sequencing and expression analysis of Dicentrarchus labrax heme oxygenase-1.

The liver cDNA encoding heme oxygenase--1 (HO-1) was sequenced from European sea bass (Dicentrarchus labrax) (accession number no. EF139130). The HO-1 cDNA was 1250 bp in nucleotide length and the open reading frame encoded 277 amino acid residues. The deduced amino acid sequence of the European sea bass had 75% and 50% identity with the amino acid sequences of tetraodontiformes (Tetraodon nigroviridis and Takifugu rubripes) and human HO-1 proteins, respectively. A short hydrophobic transmembrane domain at the C--terminal region was found, and four histidine residues were highly conserved, including human his25 that is essential for HO catalytic activity. RT-PCR of mRNA from eight different European sea bass tissues revealed that, in a homeostatis state, the heme oxygenase--1 was abundant in the spleen and liver but not in the brain.

Gene therapeutic approaches-transfer in vivo.

Osteoarthritis (OA) is common, debilitating, expensive, incurable and very difficult to treat. Gene transfer to the synovial linings of affected joints is a promising strategy for achieving sustained, therapeutic, intraarticular concentrations of anti-arthritic gene products. This is not reasonably possible with existing, alternative technologies. The present review summarizes progress in achieving direct, in vivo intraarticular gene delivery and expression. Numerous non-viral vectors have been evaluated for their ability to transfect the synovia of experimental animals following intraarticular injection. None have given more than low levels of temporary transgene expression and many are inflammatory. Several viral vectors, however, are very effective in this regard and successfully treat experimental models of OA. Adeno-associated virus has been used in a phase I study for the gene therapy of rheumatoid arthritis. Its use in a clinical trial for treating OA is pending.

Adenovirus-mediated gene transfer of glutamine: fructose-6-phosphate amidotransferase antagonizes the effects of interleukin-1beta on rat chondrocytes.

OBJECTIVE: To determine whether overexpression of glutamine: fructose-6-phosphate amidotransferase (GFAT) in synoviocytes will antagonize the response to interleukin-1beta (IL-1beta) of chondrocytes and synovial fibroblasts in co-culture. METHODS: Synovial fibroblasts from the rat were transduced by an adenovirus carrying the cDNA for GFAT and then co-cultured with rat chondrocytes encapsulated in alginate beads. Following challenge with 1, 5, or 10 ng/ml of IL-1beta for 24 h, proteoglycan synthesis by the chondrocytes was determined by incorporation of Na2(35)SO4. Production of nitric oxide (NO) and prostaglandin E2 (PGE2) were monitored by assay of conditioned medium from the co-culture. RESULTS: IL-1beta treatment of untransduced-synoviocyte/chondrocyte co-cultures resulted in markedly decreased proteoglycan synthesis by the chondrocytes, and increased NO and PGE2 levels in the culture medium. In contrast, adenovirus-mediated transfer of GFAT in synoviocytes prevented both the decrease in chondrocyte proteoglycan synthesis and increases in NO and PGE2 provoked by IL-1beta. CONCLUSIONS: Our study suggests that in a synoviocyte/chondrocyte co-culture system, overexpression of GFAT by synoviocytes significantly inhibits subsequent stimulation by IL-1beta in vitro. Since GFAT is the rate limiting enzyme in the synthesis of intracellular glucosamine and its derivatives, these results may open new possibilities for osteoarthritis treatment.

Gene delivery to cartilage defects using coagulated bone marrow aspirate.

The long-term goal of the present study is to develop a clinically applicable approach to enhance natural repair mechanisms within cartilage lesions by targeting bone marrow-derived cells for genetic modification. To determine if bone marrow-derived cells infiltrating osteochondral defects could be transduced in situ, we implanted collagen-glycosaminoglycan (CG) matrices preloaded with adenoviral vectors containing various marker genes into lesions surgically generated in rabbit femoral condyles. Analysis of the recovered implants showed transgenic expression up to 21 days; however, a considerable portion was found in the synovial lining, indicating leakage of the vector and/or transduced cells from the matrix. As an alternative medium for gene delivery, we investigated the feasibility of using coagulated bone marrow aspirates. Mixture of an adenoviral suspension with the fluid phase of freshly aspirated bone marrow resulted in uniform dispersion of the vector throughout, and levels of transgenic expression in direct proportion to the density of nucleated cells in the ensuing clot. Furthermore, cultures of mesenchymal progenitor cells, previously transduced ex vivo with recombinant adenovirus, were readily incorporated into the coagulate when mixed with fresh aspirate. These vector-seeded and cell-seeded bone marrow clots were found to maintain their structural integrity following extensive culture and maintained transgenic expression in this manner for several weeks. When used in place of the CG matrix as a gene delivery vehicle in vivo, genetically modified bone marrow clots were able to generate similarly high levels of transgenic expression in osteochondral defects with better containment of the vector within the defect. Our results suggest that coagulates formed from aspirated bone marrow may be useful as a means of gene delivery to cartilage and perhaps other musculoskeletal tissues. Cells within the fluid can be readily modified with an adenoviral vector, and the matrix formed from the clot is completely natural, native to the host and is the fundamental platform on which healing and repair of mesenchymal tissues is based.

Osteoarthritis gene therapy.

Osteoarthritis (OA) is the Western world's leading cause of disability. It is incurable, costly and responds poorly to treatment. This review discusses strategies for treating OA by gene therapy. As OA affects a limited number of weight-bearing joints and has no major extra-articular manifestations, it is well suited to local, intra-articular gene therapy. Possible intra-articular sites of gene transfer include the synovium and the cartilage. Most experimental progress has been made with gene transfer to synovium, a tissue amenable to genetic modification by a variety of vectors, using both in vivo and ex vivo protocols. The focus so far has been upon the transfer of genes whose products enhance synthesis of the cartilaginous matrix, or inhibit its breakdown, although there is certainly room for alternative targets. It is possible to build a convincing case implicating interleukin-1 (IL-1) as a key mediator of cartilage loss in OA, and the therapeutic effects of IL-1 receptor anatagonist (IL-1Ra) gene transfer have been confirmed in three different experimental models of OA. As transfer of IL-1Ra cDNA to human arthritic joints has already been accomplished safely, we argue that clinical studies of intra-articular IL-1Ra gene transfer in OA are indicated and should be funded. Of the available vector systems, recombinant adeno-associated virus may provide the best combination of safety with in vivo delivery using current technology.

Sequence protein alignment with composition new evolutions (SPACne): a program for the identification of polypeptides using amino acid composition. A user friendly modification of SPAC.

SPAC (sequence protein alignment with composition) is a software that retrieve from protein or nucleic acid databases, the sequences corresponding to a protein or peptide whose only amino acid composition and molecular weight are known. By accurately matching a DNA or a protein sequence to candidate protein or peptide fragment, this software may be used as a fast and cheap method for protein characterization. This paper describes a modification of the SPAC software, SPAC new evolutions (SPACne), which enables a more efficient and user friendly method of protein identification using amino acids composition. SPACne is available online at the web site: http://bioweb.pasteur.fr/seqanal/interfaces/spacne.html.

Glucosamine modulates IL-1-induced activation of rat chondrocytes at a receptor level, and by inhibiting the NF-kappa B pathway.

We recently reported that glucosamine reversed the decrease in proteoglycan synthesis and in UDP-glucuronosyltransferase I mRNA expression induced by interleukin-1 beta (IL-1 beta) [Arthritis Rheum. 44 (2001) 351-360]. In the present work, we show that glucosamine does not exert the same effects when chondrocytes were stimulated with reactive oxygen species (ROS). In order to better understand its mechanism of action, we determined if glucosamine could prevent the binding of IL-1 beta to its cellular receptors or could interfere with its signaling pathway at a post-receptor level. Addition of glucosamine to rat chondrocytes treated with IL-1 beta or with ROS decreased the activation of the nuclear factor kappa B, but not the activator protein-1. After treatment with IL-1 beta, glucosamine increased the expression of mRNA encoding the type II IL-1 beta receptor. These results emphasize the potential role of two regulating proteins of the IL-1 beta signaling pathway that could account for the beneficial effect of glucosamine in osteoarthritis.

Gene therapy for rheumatoid arthritis.

Rheumatoid arthritis (RA) is a disabling, painful disorder affecting 1% of the world's population. Although the aetiology of RA remains unknown, recent advances in understanding its pathophysiology have led to the characterisation of several proteins whose activities may be anti-arthritic. Clinical application of such proteins has greatly improved the treatment of RA, but the disease remains incurable and difficult to manage in a substantial number of patients. Thus, there are continued efforts to develop new therapeutic strategies. Because RA is a chronic condition, effective treatment will probably require the presence of therapeutic agents for extended periods of time. In the case of proteins, this is problematic. Gene therapy may offer a solution to this problem. Experimental studies have confirmed the feasibility, efficacy and safety of gene therapy for the treatment of animal models of arthritis. Several different approaches have shown promise in this regard, including gene transfer to the synovial lining cells of individual joints and the systemic delivery of genes to extra-articular locations. One unexpected finding has been the 'contralateral effect' in which gene delivery to one joint of an animal with polyarticular disease leads to improvement of multiple joints. Investigation of this phenomenon has led to interest in cell trafficking and the genetic modification of antigen-presenting cells (APC). The first Phase I clinical trial tested the feasibility and safety of ex vivo gene transfer to the synovial lining of human joints. This clinical trial has been successfully completed and two other Phase I trials are in progress. A Phase II study is now being planned to investigate the efficacy of gene transfer to the joints of patients with early stage RA.

Interleukin-1beta down-regulates the expression of glucuronosyltransferase I, a key enzyme priming glycosaminoglycan biosynthesis: influence of glucosamine on interleukin-1beta-mediated effects in rat chondrocytes.

OBJECTIVE: To assess the variations of galactose-beta-1,3-glucuronosyltransferase I (GlcAT-I) expression related to the decrease in proteoglycan synthesis mediated by interleukin-1beta (IL-1beta) in rat chondrocytes, and to evaluate the influence of glucosamine on the effects elicited by this proinflammatory cytokine. METHODS: Rat articular chondrocytes in primary monolayer cultures or encapsulated into alginate beads were treated with recombinant IL-1beta in the absence or presence (1.0-4.5 gm/liter) of glucosamine. Variations of GlcAT-I and expression of stromelysin 1 (matrix metalloproteinase 3 [MMP-3]) messenger RNA (mRNA) were evaluated by quantitative multistandard reverse transcriptase-polymerase chain reaction. In vitro enzymatic activity of GlcAT-I was measured by thin-layer chromatography, with radiolabeled UDP-glucuronic acid and a digalactoside derivative as substrates. Proteoglycan synthesis was determined by ex vivo incorporation of Na2-35SO4. Nitric oxide synthase and cyclooxygenase activities were monitored by the evaluation of nitrite (NO2-) and prostaglandin E2 (PGE2) produced in the culture medium, respectively. RESULTS: IL-1beta treatment resulted in a marked inhibition of GlcAT-I mRNA expression and in vitro catalytic activity, together with a decrease in proteoglycan synthesis. In addition, glucosamine was able to prevent, in a dose-dependent manner, the inhibitory effects of IL-1beta. In the same way, the amino sugar reduced NO2- and PGE2 production induced by IL-1beta. Finally, the up-regulation of stromelysin 1 (MMP-3) mRNA expression by IL-1beta was fully prevented by glucosamine. CONCLUSION: The results of this study suggest that the deleterious effect of IL-1beta on the anabolism of proteoglycan could involve the repression of GlcAT-I, a key enzyme in the biosynthesis of glycosaminoglycan. Glucosamine was highly effective in preventing these IL-1beta-mediated suppressive effects. The amino sugar also prevented the production of inflammatory mediators induced by the cytokine. This action could account for a possible beneficial effect of glucosamine on osteoarthritic articular cartilage.

Gene therapy for rheumatoid arthritis.

Advances in understanding the biology of rheumatoid arthritis (RA) have opened new therapeutic avenues. One of these, gene therapy, involves the delivery to patients of genes encoding anti-arthritic proteins. This approach has shown efficacy in animal models of RA, and the first human, phase I trial has just been successfully completed. Hand surgery featured prominently in this pioneering study, as a potentially anti-arthritic gene encoding the interleukin-1 receptor antagonist was transferred to the metacarpophalangeal joints of subjects with RA one week before total joint arthroplasty. This study has confirmed that it is possible to transfer genes safely to human joints. It should pave the way for additional application of gene therapy to arthritis and other orthopaedic conditions.

Evidence for the presence of peroxisome proliferator-activated receptor (PPAR) alpha and gamma and retinoid Z receptor in cartilage. PPARgamma activation modulates the effects of interleukin-1beta on rat chondrocytes.

Peroxisome proliferator-activated receptor (PPAR) alpha, PPARgamma, and retinoid acid receptor-related orphan receptor (ROR) alpha are members of the nuclear receptor superfamily of ligand-activated transcription factors. Although they play a key role in adipocyte differentiation, lipid metabolism, or glucose homeostasis regulation, recent studies suggested that they might be involved in the inflammation control and especially in the modulation of the cytokine production. This strongly suggests that these transcriptional factors could modulate the deleterious effects of interleukin-1 (IL-1) on cartilage. However, to date, their presence in cartilage has never been investigated. By quantitative reverse transcription-polymerase chain reaction, Western blot, and immunocytochemistry analysis, we demonstrated, for the first time, the presence of PPARalpha, PPARgamma, and RORalpha in rat cartilage, at both mRNA and protein levels. Comparatively, the PPARalpha mRNA content in cartilage was much lower than in the liver but not significantly different to that of the adipose tissue. PPARgamma mRNA expression in cartilage was weak, when compared with adipose tissue, but similar to that found in the liver. RORalpha mRNA levels were similar in the three tissues. mRNA expression of the three nuclear receptors was very differently modulated by IL-1 or mono-iodoacetate treatments. This indicates that they should be unequally involved in the effects of IL-1 on chondrocyte, which is in accordance with results obtained in other cell types. Indeed, we showed that 15d-PGJ2 mainly, but also the drug troglitazone, that are ligands of PPARgamma could significantly counteract the decrease in proteoglycan synthesis and NO production induced by IL-1. By contrast, PPARalpha ligands such as Wy-14,643 or clofibrate had no effect on this process. Therefore, the presence of PPARgamma in chondrocytes opens up new perspectives to modulate the effects of cytokines on cartilage by the use of specific ligands. The function of the two other transcription factors, PPARalpha and RORalpha identified in chondrocytes remains to be explored.

Characterization of the UDP-glucuronosyltransferases involved in the glucuronidation of an antithrombotic thioxyloside in rat and humans.

To investigate the glucuronidation on the hydroxyl group of carbohydrate-containing drugs, the in vitro formation of glucuronides on the thioxyloside ring of the antithrombotic drug, LF 4.0212, was followed in rat and human liver microsomes and with recombinant UDP-glucuronosyltransferases (UGT). The reaction revealed a marked regioselectivity in rat and humans. Human liver microsomes glucuronidated the compound mainly on the 2-hydroxyl position of the thioxyloside ring, whereas rat was able to form glucuronide on either the 2-, 3-, or 4- hydroxyl group of the molecule, although to a lower extent. LF 4.0212 was a much better substrate of human UGT than the rat enzyme (Vmax/Km 30.0 and 0.06 microl/min/mg, respectively). Phenobarbital, 3-methylcholanthrene, and clofibrate enhanced the glucuronidation of LF 4.0212 on positions 2, 3, and 4 of the thioxyloside ring, thus indicating that several UGT isoforms were involved in this process. The biosynthesis of the 2-O-glucuronide isomer was catalyzed by the human UGT1A9 and 2B4, but not by UGT1A6 and 2B11. By contrast, the rat liver recombinant UGT1A6 and 2B1 failed to form the 2-O-glucuronide isomers. From all the recombinant UGTs tested, none catalyzed the formation of the 3-O-glucuronide isomer. Interestingly, glucuronidation on the 4-position was found in all the metabolic competent V79 cell lines considered, including the nontransfected V79 cells, suggesting the presence of an endogenous UGT in fibroblasts able to actively glucuronidate the drug. This activity, which was nonsensitive to the inhibitory effect of 7,7,7-triphenylheptanoic acid, a potent UGT inhibitor, could reflect the existence of a different enzyme.