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The chondrocyte is the unique cell type in articular cartilage. It is responsible for the extracellular matrix production which permits the cartilaginous tissue to compensate the skeletal pressures during movements. The regulation of the different extracellular matrix components (type II collagen and aggrecan) depends on the differentiated state of the chondrocyte. However, in vivo, such as during aging and osteoarthritis, as well as in vitro, in monolayer culture, chondrocytes lose their morphological and biochemical characteristics. This phenomenon has been particularly studied in culture. As soon as at passage 1, there is a gradual shift from the synthesis of type II collagen to type I and III collagens and from the synthesis of large aggregating proteoglycans (aggrecan) to low molecular weight proteoglycans. However, it has been shown that dedifferentiated chondrocytes can reexpress phenotypic markers of the articular chondrocyte. These conditions include tridimensional culture in gels of agarose, collagen and alginate. This can be also obtained after treatment of chondrocytes in monolayer by dihydrocytochalasin B or staurosporine. Although the mechanisms involved in restoration of the differentiated phenotype have not been elucidated yet, it has been shown that the synthesis of specific proteoglycans and type II collagen can be related to a modification of actin architecture. The restoration of the differentiated functions using tridimensional culture of chondrocytes has been used to perform autologous chondrocyte implantation in chondral defects. In the future, the grafts could be improved using chondrocytes treated with stimulating growth factors and synthetically derived matrices.

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Chondrocytes cultivated in monolayer rapidly divide and lose their morphological and biochemical characteristics, whereas they maintain their phenotype for long periods of time when they are cultivated in alginate beads. Because cartilage has a low cellularity and is difficult to obtain in large quantities, the number of available cells often becomes a limiting factor in studies of chondrocyte biology. Therefore, we explored the possibility of restoring the differentiated properties of chondrocytes by cultivating them in alginate beads after two multiplication passages in monolayer. This resulted in the reexpression of the two main markers of differentiated chondrocytes: Aggrecan and type II collagen gene expression was strongly reinduced from day 4 after alginate inclusion and paralleled protein expression. However, 2 weeks were necessary for total suppression of type I and III collagen synthesis, indicators of a modulated phenotype. Interleukin-1beta, a cytokine that is present in the synovial fluid of rheumatoid arthritis patients, induces many metabolic changes on the chondrocyte biology. Compared with cells in primary culture, the production of nitric oxide and 92-kDa gelatinase in response to interleukin-1beta was impaired in cells at passage 2 in monolayer but was fully recovered after their culture in alginate beads for 2 weeks. This suggests that the effects of interleukin-1beta on cartilage depend on the differentiation state of chondrocytes. This makes the culture in alginate beads a relevant model for the study of chondrocyte biology in the presence of interleukin-1beta and other mediators of cartilage destruction in rheumatoid arthritis and osteoarthrosis.

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The purpose of this study was to investigate the implication of transglutaminases in the biology of articular chondrocytes. Transglutaminase activity measurements performed on cell lysates showed that a transglutaminase was present in chondrocytes in primary culture and that it was strongly activated by limited proteolysis. In chondrocytes dedifferentiated by subculture or retinoic acid treatment, this transglutaminase appeared to be downregulated, while type II transglutaminase expression was induced. However, protein levels, mRNA steady-state levels or transglutaminase activity in whole-cell lysates do not necessarily reflect the activity present in living cells, as it is strongly regulated. Therefore, Fluoresceincadaverine, a fluorescent polyamine, was used for detecting amine acceptor protein substrates accessible to active transglutaminase in living cells. After incubation of chondrocytes with Fluoresceincadaverine, dedifferentiated cells exhibited an extracellular labelling, while chondrocytes in primary culture did not, unless thrombin was added to the culture medium. In contrast, Fluoresceincadaverine labelling was not detected in the cytosol, although the transglutaminases were also partly cytosolic. By confocal microscopy and Western blot analysis of labelled cells in culture, fibronectin was shown to be the main substrate for both transglutaminases. The transglutaminases present in articular chondrocytes may, therefore, contribute to the organization and the stabilization of their extracellular matrix.

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Among the various directions explored in order to have a large number of differentiated articular chondrocytes easily available, the restoration of the differentiated properties after cell multiplication in monolayer has been proposed. It has been clearly shown that the synthesis of cartilage proteoglycans and type II collagen synthesis is coincident with the presence of a faint microfibrillar architecture but is absent in chondrocytes showing well-defined actin cables. Staurosporin, mainly described as a protein kinase C inhibitor, has also been shown to rapidly induce the disruption of the actin microfilaments. The purpose of this paper was to investigate whether properties of differentiated chondrocytes were reinitiated upon staurosporin treatment of serially passaged chondrocytes. Results showed, after staurosporine treatment of cells at Passage two for 5 d, complete suppression of type I and type III collagen synthesis and induction of type II collagen synthesis and of Alcian blue stainable matrix. Additionally, we showed that staurosporin restored metabolic responses that chondrocytes in primary culture exhibit upon interleukin-1 beta treatment (decrease of Alcian blue- positive cells, induction of expression of the 92 kDa gelatinase, nitric oxide production). We conclude that staurosporin is a potent redifferentiating agent of articular chondrocytes that have been subcultured up to Passage two for multiplication. Taking into account that the cellularity of cartilage is very low, staurosporine-treated chondrocytes could be useful as an alternative cellular model to evaluate pharmacotoxicological effects of drugs.

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The use of Fluoresceincadaverine as a primary amine donor for detecting the endogenous substrates for active transglutaminase in living cells was studied. Fluoresceincadaverine was found to be suitable for labelling cells in culture as it did not induce cytotoxicity when used at 0.5 mM in culture media and diffused throughout the cell. After appropriate fixation using methanol, Fluoresceincadaverine-labelled cells were observed by direct fluorescence microscopy, allowing visualization of the substrates for active transglutaminase. Simultaneous detection of transglutaminase and of Fluoresceincadaverine incorporated into proteins strongly suggested that cytosolic transglutaminase was inactive in these living cells. However, transglutaminase co-distributed with Fluoresceincadaverine-labelled structures, which resembled a lattice. Fluoresceincadaverine-labelled proteins detected by Western blotting using an anti-Fluorescein antibody showed that, in living cells, the major transglutaminase substrate migrated at an apparent molecular weight of 220 kDa, as does fibronectin. Fibronectin was found to co-distribute with Fluoresceincadaverine-labelled lattice. This confirmed that these lattice structures were extracellular and, therefore, that transglutaminase is in an active form in this compartment. This opportunity to perform morphological and biochemical analyses in the search for transglutaminase substrates in living cells should help in determining the specific function of transglutaminases in a particular cell type as well as in universal cellular events, such as apoptosis or cell growth.

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Depending on the cell type studied, the involvement of type II transglutaminase (TGase) has been proposed in almost any event of the cell life such as differentiation, apoptosis, growth, aging, cell morphology and adhesion, metastatic capacity or extracellular matrix stabilization. In order to define the field(s) where this enzyme may be implicated in chondrocytes, type II TGase expression was studied in chondrocytes at different passages which differentiated state was modulated by retinoic acid, dihydrocytochalasin B or staurosporin. Results showed that (i) type II TGase expression is not incompatible with type II collagen expression, a main marker of chondrocyte differentiation (ii) type II TGase expression is higher when cells are in the exponential phase of growth than when growth arrested (iii) a high type II TGase expression does not imply that cells are apoptotic although cell apoptosis correlates with increased type II TGase expression (iv) non-adherent cells do not express type II TGase whereas adherent cells do whatever their differentiation state as assessed by type II collagen synthesis. These results suggest that, in articular chondrocytes, type II TGase is specifically implicated in the cell adhesion capacity.

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In vivo, articular chondrocytes produce an important amount of extracellular matrix (cartilage) whose quality is impaired upon inflammation or aging leading to arthritis or arthrosis. Transglutaminases (EC 2.3.2.13) are a family of enzymes which have been shown to be involved in extracellular matrix stabilization, cell differentiation and possibly in initiation and propagation of inflammatory diseases. It is therefore of interest to study transglutaminase activity in chondrocytes. Transglutaminase activity was studied in rabbit articular chondrocytes in primary culture, where cells are in a well-differentiated state as assessed by collagen-type synthesis, as well as in subculture and in retinoic acid-treated cells, where cells are in a dedifferentiated state. Results showed that two different TGases activities are expressed in chondrocytes. One, down-regulated upon retinoic acid treatment of cells, preferentially membrane bound and strongly activated upon trypsin treatment of cell lysates, is expressed at a high level in primary culture. The other one is up-regulated upon retinoic acid treatment, preferentially cytosolic and inactivated upon trypsin treatment of cell lysates. The rate of expression of the TGase down-regulated by RA seems to correlate with the differentiation state of the chondrocyte. This suggests that this TGase activity may have a physiological role in cartilage and merits further study.

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Macromolecules (substitutive enzymes, polymeric prodrugs, immunotoxins, radiolabeled antibodies, or peptide hormones) are of interest in the treatment of several diseases. To reach the tissues, these macromolecular drugs have to cross the capillary wall, which represents an important transfer limitation. While pharmacokinetics usually studies the changes in drug concentration in different body compartments, analyzing the amount of drug gaining access to its target may be more relevant for assessing the efficiency of macromolecules than for low molecular mass drugs. To determine the influence of different parameters on the fraction of the injected dose gaining access to the pharmacologic target, we constructed pharmacokinetic models where two uptakes, both linear or nonlinear, work either in the same compartment (no transport limitation), or in compartments separated by a transport barrier. Numerical applications were carried out with parameters obtained either experimentally or from the literature. We conclude that it is of little use to increase the affinity (K(uptake)) of a macromolecular drug for its target when a transport limitation and an undesired elimination from the plasma space are both present. Likewise, an increase of the uptake (rate of uptake or maximal velocity) by the target is not very productive because permeability of the capillary wall is the factor limiting access of macromolecules to tissues. Maximal efficiency of therapeutic macromolecules could be achieved by increasing, where feasible, the transport across the barrier between the plasma and the target, and by preventing the undesired eliminations as much as possible.

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Human articular cartilage cells were transfected with the t.-sensitive polyomavirus large T antigen of SV40. Several immortalized chondrocyte cell lines were obtained. The types of acidic polysaccharides and of collagen synthesized suggest dedifferentiation in the in vitro culture system used afterwards to obtain large numbers of cells.

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The effect of syngeneic anti-idiotypic (anti-id) antibodies on the biodistribution of three murine monoclonal antibodies (mAb) against human tumour-associated antigens, and also on that of their fragments, has been examined in mice using, as a model system, purified anti-id mAb against three different target mAb. With the IgG2b mAb NCRC-2, pretreatment of mice 24 h previously with its IgG1 anti-id mAb NCRC-60 caused clearance of subsequently administered NCRC-2. With the univalent Fab/c fragment of NCRC-2 there was little effect, even with anti-id to Fab/c pretreatment ratios of 20:1, although immune complexes were present in the circulation. With Fab of NCRC-2, anti-id mAb prolonged blood survival by reducing renal clearance, immune complexes surviving in the circulation. With the IgG1 mAb NCRC-23, immune complexes formed in vivo with the IgG2b anti-id mAb NCRC-59, but with only little hepatic clearance. With the Fab and F(ab')2 fragments of NCRC-23, blood survival was increased in mice pretreated with anti-id mAb, and with Fab this was clearly due to reduced renal clearance. The third mAb, the IgG3 NCRC-48, formed complexes with its IgG2a anti-id mAb NCRC-62, but these survived in the circulation with no accelerated clearance of the target mAb. These results are different from those previously seen with endogenous anti-id responses. They indicate the diversity of effects that anti-id mAb can have on the biodistribution of their target mAb, and emphasise the difficulty of using such anti-id mAb to modulate the pharmacokinetics of target mAb.

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Cell lines were established from rabbit articular chondrocytes following transfection with a plasmid encoding SV40 early function genes. This resulted in cell immortalization (130 passages have been completed for the oldest cell line) with acquisition of characteristics of partial transformation such as reduced serum requirements for normal and clonal growth. The immortalized chondrocytes, called SVRAC, did not form multilayer foci when maintained in postconfluent culture. Their ability to form colonies in soft agar was not increased in comparison with normal chondrocytes, but they were weakly tumorigenic in nude mice. SVRAC lost the ability to synthesize type II collagen and Alcian blue-stainable matrix, which are markers of the differentiated chondrocyte phenotype, and synthesized predominantly type I collagen. Studies of collagen gene expression showed that pro alpha 1 (II) mRNA was undetectable, whereas pro alpha 1 (I) collagen mRNA was expressed even in late passage cultures. Unlike normal dedifferentiated chondrocytes, SVRAC were unable to re-express the differentiated phenotype in response to tridimensional culture or microfilament depolymerization. Cell lines obtained from chondrocytes transfected either in primary culture or just after release of cells from cartilage displayed the same behaviour. Thus SV40 early genes were able to immortalize rabbit articular chondrocytes, but the resulting cell lines displayed an apparently irreversibly dedifferentiated phenotype. These cell lines can be used as models to identify regulatory pathways that are required for the maintenance or reexpression of differentiated function in chondrocytes.

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Radioiodine-labelled 791T/36 monoclonal antibody (mAb) and its Fab/c fragment, consisting of one Fab arm and the Fc portion, have identical whole-body survival curves in BALB/c mice (t1/2 = 3.75 days). Therefore, these two forms of this antibody provide a suitable model for studying the role of valency in the targeting efficiency of antibodies to tumours in vivo. 791/T36 antibody and its Fab/c fragment were labelled either by direct iodination using the iodogen method (125I) or by dilactitol-125I-tyramine (125I-DLT), a residualizing label, which accumulates in the cells involved in degradation of the carrier protein. In tumour-bearing nude mice, the percentage of injected dose of mAb or Fab/c fragment reaching the specific 791T tumour was similar, and these proteins appeared to be catabolized at a similar rate in this tissue. mAb, but not the Fab/c fragment, was found to be very actively catabolized by the liver and spleen of tumour-bearing mice compared to control nude mice, this probably resulting from clearance of immune complexes. This effect was most pronounced when the mAb was labelled with 125I-DLT, the percentage of injected dose of mAb reaching the spleen and liver being higher than the percentage of injected dose reaching the tumour. This effect was not seen with the Fab/c fragment. Autoradiographic studies on tumour sections, which exhibit antigenic sites throughout the tumour mass, showed that the Fab/c fragment was already homogeneously distributed in the tumour 12 h after injection whereas the whole antibody was mainly localized at the periphery of the tumour. Those results suggest a "binding site barrier" effect. Overall, these results indicate that the highest valency and affinity may not be the optimal choice for mAb to be used for therapeutic purposes.

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The articular cartilage located in movable joints can be divided into cellular and intercellular components. This latter category, also called the extracellular matrix, is responsible for the mechanical properties of cartilage and results from the functional activity of the cartilaginous cells, the chondrocytes. Through changes in their metabolism, chondrocytes are able to modify the composition of cartilage in response to physiological, pathological and pharmacotoxicological stimuli. The culture of articular chondrocytes is therefore important in biological research and various methods have been proposed. Models in vitro of normal and pathological chondrocytes are now described. Normal culture can be studied in organ explants, monolayers and various types of tridimensional culture. Chondrocytes immortalized by gene transfection will provide a further model of normal cells when progress has been made in the maintenance of differentiation. Models of pathological cells involve normal cultured chondrocytes treated with cytokines or oxygen free radicals, or rendered senescent by successive passages. Such models are useful in pharmacological and toxicological research. The advantages and limitations of these different models of cultured articular chondrocytes are discussed.

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Fab/c fragments, purified from pepsin digest of mouse IgG2b monoclonal antibody (MoAb) 791T/36, consist of one Fab arm and the intact Fc portion. Pharmacokinetic and biodistribution studies in BALB/c mice of radioiodine-labeled 791T/36 MoAb and its Fab/c fragments showed that, due to the presence of the Fc portion, the Fab/c fragment has the same catabolic rate as whole antibody (T1/2 = 64 h). Due to its lower molecular weight (105,000), the Fab/c fragment extravasated more quickly and to a greater extent than whole MoAb in organs in which the vascular endothelium was fenestrated or continuous. In organs in which the vascular endothelium is sinusoidal, such as in liver and spleen, their diffusion capacities were identical. Therefore, Fab/c fragments reconcile advantages of the intact antibody molecule (slow catabolic rate) and Fab or F(ab)2 fragments (increased extravascular diffusion), features required to improve targeting to solid tumors. Data from biodistribution studies in nude mice bearing subcutaneous 791T tumor (antigen positive) and Colo205 tumor (antigen negative) contralaterally showed important differences in the behavior of whole MoAb and Fab/c fragment: (a) Whole MoAb was cleared more rapidly from the body and from the blood than Fab/c fragment; (b) The MoAb was taken up by the spleen (tissue to blood ratio greater than 1 from 12 h after injection over the 3 days of the experiment) and the liver (0.6), whereas Fab/c fragment tissue to blood ratios were only slightly increased (0.34 and 0.35) compared to control nude mice (0.25 and 0.28) for the spleen and liver, respectively, 3 days after injection. Since both MoAb and Fab/c fragment bear the Fc portion, these data suggest that the reputed "nonspecific uptake" of antibodies due to the Fc portion could be an Fc-mediated specific uptake, e.g., uptake of immune complexes; (c) The tumor to blood ratios were 1.7 and 1.2 for MoAb and Fab/c fragment, respectively, from 24 h throughout the experiment, whereas the percentage of injected dose (% of ID) present/g of 791T tumor was at any time greater for Fab/c fragment (8% maximum of ID) than for MoAb (5% of ID). These results were not expected in view of the low immunoreactivity in vitro of Fab/c fragments compared to whole antibody. It is suggested that the distribution and the catabolic rate of whole antibody and its Fab/c fragment at the tumor level are modulated by their respective valency and immunoreactivity for the target cell.

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Mouse IgG2b fragmentation has been poorly described in the literature because of the sensitivity of this subclass to proteases and the inability to produce F(ab')2 fragments. The fragments obtained include both Fc and Fab fragments and an intermediate of degradation, the Fab/c fragment, consisting of the Fc region and one Fab arm, which was first described by Parham (1983). Optimised pepsin digestion led to the formation of Fab/c in yields of up to 30% depending on the IgG2b antibodies susceptibility. DEAE-cellulose chromatography of the digested antibodies allowed, in all cases, separation of Fab fragments from Fc bearing fragments. Depending on the differences in pI between the fragments, Fab/c fragment purification was achieved either by CM-cellulose chromatography or by recycling HPLC gel filtration chromatography. Both procedures gave 97.5% purity Fab/c fragments. Fc fragments were purified by HPLC gel filtration chromatography. In cancer therapy the monovalent Fab/c fragments could be useful for drug targeting or for immunotherapy providing it retains a good affinity.

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High background staining due to glutaraldehyde fixation prevents phenazine methosulphate and a tetrazolium salt being used to visualize glucose oxidase activity in tissue slices prepared from mice injected with the enzyme. Experiments in solution showed that products formed during the reaction between amino groups and glutaraldehyde are, at least in part, responsible for the non-enzymatic reduction of tetrazolium salts. Experiments performed with artificial membranes chemically akin to glutaraldehyde-fixed sections and prepared by cross-linking albumin by glutaraldehyde, showed that double bonds in amino-glutaraldehyde products are mainly responsible for the background staining development, whereas thiol groups play only a minor role. A sequential treatment with sodium borohydride and N-ethylmaleimide greatly reduced the background staining, thus permitting the detection of glucose oxidase activity. Optimal conditions for glucose oxidase activity demonstration (maximum enzyme velocity for minimum 'nothing dehydrogenase' phenomenon) were studied: choice of the tetrazolium salt, nature, pH and molarity of the buffer used for the staining mixture. A procedure similar to that developed with artificial membranes was applied to tissue sections of mice in which glucose oxidase had been injected intravenously. It allowed detection of glucose oxidase activity without artifactual staining in control slices.

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The administration of enzymes is of potential therapeutic value in many disease states, e.g. lysosomal storage diseases, provided problems in the metabolism and targeting of large proteins can be overcome. We have addressed ourselves to these problems by studying the pharmacokinetics and distribution of glucose-oxidase (GO) and some of its derivatives in mice. A saturable mechanism was responsible for GO uptake by mononuclear phagocytes. After construction of a pharmacokinetic model, the Kuptake (850 nmol/l) and the number of capturing cells were determined; uptake was half the initial plasma concentration in about 10 min. Deglycosylated GO's had half-lives of about 100 min and were taken up by the same organs that took up native GO. Galactosylated GO had a half-life of 4 min and a different distribution; it was taken up preferentially by the liver in hepatocytes. Our results illustrate the role sugars might play in the targeting of foreign proteins to different cell types, and the feasibility of determining in vivo microscopic constants such as the affinity between molecules and certain cells.

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