Platelet-released growth factors can accelerate tenocyte proliferation and activate the anti-oxidant response element.

Little is know about the pathophysiology of acute and degenerative tendon injuries. Although most lesions are uncomplicated, treatment is long and unsatisfactory in a considerable number of cases. Besides the common growth factors that were shown to be relevant for tendon integrity more recently protection against oxidative stress was shown to promote tendon healing. To improve tendon regeneration, many have advocated the use of platelet-rich plasma (PRP), a thrombocyte concentrate that can serve as an autologous source of growth factors. In this study, we investigated the effect of platelet-released growth factors (PRGF) on tenocytes. Tenocytes were isolated from the Achilles tendon of postnatal rats. Tenocyte cell cultures were stimulated with PRGF. We used a CyQuant assay and WST assay to analyse tendon cell growth and viability in different concentrations of PRGF. Migration and proliferation of cells grown in PRGF were assessed by a scratch test. A dual-luciferase assay was used to demonstrate the activation of the anti-oxidant response element (ARE) in tenocytes. A positive effect of PRGF could be shown on tendon cell growth and migratory capacity. PRGF activated the Nrf2-ARE pathway in a dose-dependent manner. Here, we provide evidence of a biological effect of PRGF on tenocytes by the promotion of tenocyte growth and activation of the Nrf2-ARE pathway. This is a novel aspect of the action of platelet concentrates on tendon growth.

Dual interaction of the malaria circumsporozoite protein with the low density lipoprotein receptor-related protein (LRP) and heparan sulfate proteoglycans.

Speed and selectivity of hepatocyte invasion by malaria sporozoites have suggested a receptor-mediated mechanism and the specific interaction of the circumsporozoite (CS) protein with liver-specific heparan sulfate proteoglycans (HSPGs) has been implicated in the targeting to the liver. Here we show that the CS protein interacts not only with cell surface heparan sulfate, but also with the low density lipoprotein receptor-related protein (LRP). Binding of 125I-CS protein to purified LRP occurs with a Kd of 4.9 nM and can be inhibited by the receptor-associated protein (RAP). Blockage of LRP by RAP or anti-LRP antibodies on heparan sulfate-deficient CHO cells results in more than 90% inhibition of binding and endocytosis of recombinant CS protein. Conversely, blockage or enzymatic removal of the cell surface heparan sulfate from LRP-deficient embryonic mouse fibroblasts yields the same degree of inhibition. Heparinase-pretreatment of LRP-deficient fibroblasts or blockage of LRP on heparan sulfate-deficient CHO cells by RAP, lactoferrin, or anti-LRP antibodies reduces Plasmodium berghei invasion by 60-70%. Parasite development in heparinase-pretreated HepG2 cells is inhibited by 65% when RAP is present during sporozoite invasion. These findings suggest that malaria sporozoites utilize the interaction of the CS protein with HSPGs and LRP as the major mechanism for host cell invasion.

Biological actions of curcumin on articular chondrocytes.

OBJECTIVES: Curcumin (diferuloylmethane) is the principal biochemical component of the spice turmeric and has been shown to possess potent anti-catabolic, anti-inflammatory and antioxidant, properties. This article aims to provide a summary of the actions of curcumin on articular chondrocytes from the available literature with the use of a text-mining tool. We highlight both the potential benefits and drawbacks of using this chemopreventive agent for treating osteoarthritis (OA). We also explore the recent literature on the molecular mechanisms of curcumin mediated alterations in gene expression mediated via activator protein 1 (AP-1)/nuclear factor-kappa B (NF-kappaB) signalling in chondrocytes, osteoblasts and synovial fibroblasts. METHODS: A computer-aided search of the PubMed/Medline database aided by a text-mining tool to interrogate the ResNet Mammalian database 6.0. RESULTS: Recent work has shown that curcumin protects human chondrocytes from the catabolic actions of interleukin-1 beta (IL-1beta) including matrix metalloproteinase (MMP)-3 up-regulation, inhibition of collagen type II and down-regulation of beta1-integrin expression. Curcumin blocks IL-1beta-induced proteoglycan degradation, AP-1/NF-kappaB signalling, chondrocyte apoptosis and activation of caspase-3. CONCLUSIONS: The available data from published in vitro and in vivo studies suggest that curcumin may be a beneficial complementary treatment for OA in humans and companion animals. Nevertheless, before initiating extensive clinical trials, more basic research is required to improve its solubility, absorption and bioavailability and gain additional information about its safety and efficacy in different species. Once these obstacles have been overcome, curcumin and structurally related biochemicals may become safer and more suitable nutraceutical alternatives to the non-steroidal anti-inflammatory drugs that are currently used for the treatment of OA.

Co-culture of canine mesenchymal stem cells with primary bone-derived osteoblasts promotes osteogenic differentiation.

Tissue engineering of bone grafts with osteogenic progenitor cells such as adult mesenchymal stem cells (MSC) represents a promising strategy for the treatment of large bone defects. The aim of this experimental study was to evaluate the osteogenic potential of primary osteoblasts on MSCs in co-culture at different ratios. The co-cultures were treated with or without a specific osteogenic induction medium in monolayer and high density cultures. In monolayer co-cultures, MSCs and osteoblasts actively searched for cell-cell contact leading to cell proliferation and only in treated monolayer co-cultures osteogenesis was observed. Ultrastructural evaluation of high density co-cultures using electron microscopy demonstrated osteogenesis with no significant difference between treated or untreated co-cultures. Immunoblotting confirmed expression of collagen type I, beta1-Integrin, the osteogenic-specific transcription factor Cbfa-1 and induction of the MAPKinase pathway (Shc, Erk1/2) in both treated and untreated co-cultures. Although treatment with the induction medium enhanced osteogenesis in the co-cultures compared to untreated co-cultures, the quality of osteogenesis was proportional to the quantity of osteoblasts in the co-cultures. Fifty percent osteoblasts in the co-cultures markedly increased osteogenesis; even the presence of ten percent osteoblasts in the co-culture strongly promoted osteogenesis. This data leads us to conclude that co-culture of MSCs with osteoblasts combined with the three-dimensional environment of the high density culture strongly promotes osteogenesis and stabilizes the osteogenic potential of MSCs. This approach may prove to be of practical benefit in future tissue engineering and regenerative medicine research.

Diverse roles of integrin receptors in articular cartilage.

Integrins are heterodimeric integral membrane proteins made up of alpha and beta subunits. At least eighteen alpha and eight beta subunit genes have been described in mammals. Integrin family members are plasma membrane receptors involved in cell adhesion and active as intra- and extracellular signalling molecules in a variety of processes including embryogenesis, hemostasis, tissue repair, immune response and metastatic spread of tumour cells. Integrin beta 1 (beta1-integrin), the protein encoded by the ITGB1 gene (also known as CD29 and VLAB), is a multi-functional protein involved in cell-matrix adhesion, cell signalling, cellular defense, cell adhesion, protein binding, protein heterodimerisation and receptor-mediated activity. It is highly expressed in the human body (17.4 times higher than the average gene in the last updated revision of the human genome). The extracellular matrix (ECM) of articular cartilage is a unique environment. Interactions between chondrocytes and the ECM regulate many biological processes important to homeostasis and repair of articular cartilage, including cell attachment, growth, differentiation and survival. The beta1-integrin family of cell surface receptors appears to play a major role in mediating cell-matrix interactions that are important in regulating these fundamental processes. Chondrocyte mechanoreceptors have been proposed to incorporate beta1-integrins and mechanosensitive ion channels which link with key ECM, cytoskeletal and signalling proteins to maintain the chondrocyte phenotype, prevent chondrocyte apoptosis and regulate chondrocyte-specific gene expression. This review focuses on the expression and function of beta1-integrins in articular chondrocytes, its role in the unique biology of these cells and its distribution in cartilage.

Hypoxia inducible factor-1 and facilitative glucose transporters GLUT1 and GLUT3: putative molecular components of the oxygen and glucose sensing apparatus in articular chondrocytes.

Articular cartilage is an avascular connective tissue in which the availability of oxygen and glucose is significantly lower than synovial fluid and plasma. Glucose is an important metabolic fuel and structural precursor that plays a key role in the synthesis of extracellular matrix macromolecules in articular cartilage. However, glucose concentrations in cartilage can fluctuate depending on age, physical activity and endocrine status. Chondrocytes are glycolytic cells and must be able to sense the quantities of oxygen and glucose available to them in the extracellular matrix and respond appropriately by adjusting cellular metabolism. Consequently chondrocytes must have the capacity to survive in an extracellular matrix with limited nutrients and low oxygen tensions. The molecular mechanisms responsible for allowing chondrocytes to adapt to these harsh environmental conditions are poorly understood. In this article we present a novel "dual" model of oxygen and glucose sensing in chondrocytes based on recent experimental data. This model incorporates the hypoxia-inducible factor alpha (HIF-1alpha) as an oxygen sensor and the hypoxia responsive facilitative glucose transporters, GLUT1 and GLUT3 as putative components of the glucose sensing apparatus in chondrocytes. Recent studies have shown that GLUT1 and GLUT3 are both expressed in chondrocytes and their HIF-1alpha-mediated transcription may be dually stimulated in response to hypoxia and low glucose conditions which in turn promote anaerobic glycolysis in favor of oxidative phosphorylation. This working model provides, for the first time, a unifying hypothesis to explain how chondrocytes might sense and respond to low oxygen tensions and alterations in extracellular glucose.

Ultrastructural changes associated with myocardial apoptosis, in failing rat hearts induced by volume overload.

BACKGROUND: Myocardial apoptosis has been discussed to play a pivotal role in the development and progression of congestive heart failure (CHF). However, recently there is doubt on the evidence of myocardial apoptosis in heart failure as information on ultrastructural changes by electron microscopy is still scarce. This project therefore aimed to detect direct morphological evidence of myocardial apoptosis in an experimental heart failure model. METHOD: Following IRB approval, an aortocaval fistula (ACF) was induced in male Wistar rats using a 16G needle. 28±2days following ACF rats were examined by hemodynamic measurements, Western blot, immunofluorescence confocal and electron microscopic analysis. RESULTS: Within 28±2days of ACF heart (3.8±0.1 vs. 6.6±0.3mg/g) and lung (3.7±0.2 vs. 6.9±0.5mg/g) weight indices significantly increased in the ACF group accompanied by a restriction in systolic (LVEF: 72±2 vs. 39±3%) and diastolic (dP/dtmin.: -10,435±942 vs. -5982±745mmHg/s) function (p<0.01). Activated caspase-3 was significantly increased in failing hearts concomitant with mitochondrial leakage of cytochrome c into the cytosol. Finally, electron microscopy of the left ventricle (LV) of ACF rats revealed pronounced ultrastructural changes in >70% of examined cardiomyocytes, such as nuclear chromatin condensation, myofibril loss and disarray, contour irregularities and amorphous dense bodies, mitochondriosis and damaged cell-cell-contacts between cardiomyocytes. CONCLUSIONS: Volume overload induced heart failure is associated with activation of the mitochondrial apoptotic pathway. In addition, electron microscopy of the LV revealed direct ultrastructural evidence of extended myocardial apoptosis in ACF rats.

Co-existence of NADPH-diaphorase, fibroblast growth factor-2 and fibroblast growth factor receptor in spinal autonomic system suggests target-specific actions.

In the rat spinal cord, we found substantial co-existence of fibroblast growth factor-2, fibroblast growth factor receptor (type-1 or flg) immunoreactivity and reduced nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase activity (a histochemical marker for neuronal nitric oxide synthase) in preganglionic autonomic cell groups of intermediate layers VI, VII and X. Anti-fibroblast growth factor-2 and anti-nitric oxide synthase binding sites were confined to the cytoplasm of reactive neurons as judged by immunogold electron microscopy. Within the major autonomic nucleus, i.e. intermediolateral column, three different populations were identified: (i) fibroblast growth factor and fibroblast growth factor receptor, (ii) fibroblast growth factor/NADPH-diaphorase and (iii) NADPH-diaphorase-only stained cell groups. Sympathoadrenal neurons were prelabelled with fluorescent tracer Fast Blue and co-stained for fibroblast growth factor-like protein and NADPH-diaphorase, suggesting heterologous diversification of neuronal phenotypes and functional organization in the spinal autonomic system. Our findings suggest intriguing roles for nitric oxide and fibroblast growth factor-2 cytokine in the preganglionic sympathetic spinal cord system: The "short-term" diffusible messenger nitric oxide may act as "tonic" and/or "phasic" signal within rostrocaudally oriented function-specific preganglionic units necessary for integrated target control. The "long-term" messenger fibroblast growth factor-2 may be involved in, for example, cytokine-dependent regulation of neuronal NADPH-diaphorase/nitric oxide synthase. Furthermore, co-existence of NADPH-diaphorase, fibroblast growth factor-2 and receptor in sympathoadrenal neurons suggest mutual target-specific regulatory functions, e.g. hormone release and blood perfusion or maintenance of phenotype and plasticity responsiveness of adrenal medullary tissue.

Changes in integrin expression during chondrogenesis in vitro: an immunomorphological study.

Integrins are receptors composed of ligand-specific alpha-chains and cell type-specific beta-chains which are involved in cell-cell and cell-matrix interactions. The distribution of alpha 1- and alpha 3-integrins as well as collagen Types I and II, was investigated by immunofluorescence and immunoelectron microscopy during chondrogenesis in organ culture after various culture periods. Mesenchymal cells from limb buds of Day 12 mouse embryos were grown at high density. Within the first 2 days of the culture period, only alpha 1-integrin could be detected. Formation of cartilage-specific matrix on Day 3 was accompanied by the occurrence of alpha 3-integrin. On Day 7, alpha 3 was present only in cartilage nodules, whereas alpha 1 was strongly expressed in the perichondrium and was more or less homogeneously distributed in the surrounding mesenchyme. On Day 14, alpha 1-integrin was again detectable in cartilage. We suggest that the change in collagen formation from Type I to Type II during chondrogenesis is accompanied by a change in integrin expression from alpha 1 to alpha 3. Conversely, dedifferentiation of chondrocytes in aging cartilage is accompanied by the occurrence of collagen Type I and alpha 1-integrin. Therefore, a strict correlation between the collagen type synthesized by the cells and the appropriate receptor presented by the cells is suggested.

Integrin expression on epiphyseal mouse chondrocytes in monolayer culture.

The expression of alpha 1-, alpha 3-, alpha v- and alpha 5 beta 1-integrins and their specific ligand binding were investigated in monolayer cultures of chondrocytes from 17-day-old mouse embryos using morphological and immunomorphological methods. After a 1-day culture period alpha v-, alpha 3- and alpha 5 beta 1-integrins were observed on these cells. Immunoelectronmicroscopic investigation revealed localization predominantly in the contact areas with extracellular structures on the cell surface. alpha 1-integrin could not be demonstrated on chondrocytes. After a 5-day culture period the number of fibroblast-like cells with alpha 1-, alpha v- and alpha 5 beta 1-integrins had increased. alpha 3-integrin was hardly recognizable on these cells. Collagen type I and fibronectin could be shown as ligands on the cell surface. The number of chondrocytes with collagen type II on their surface continuously decreased. alpha 3-integrin is obviously responsible for the binding of collagen type II, and alpha 1-integrin for the binding of collagen type I. Therefore, it can be concluded that the changes of chondrocytes to fibroblast-like cells in monolayer culture are accompanied by changes of integrin genes.

Beta1-integrins co-localize with Na, K-ATPase, epithelial sodium channels (ENaC) and voltage activated calcium channels (VACC) in mechanoreceptor complexes of mouse limb-bud chondrocytes.

Interactions between chondrocytes and their extracellular matrix are partly mediated by beta1-integrin receptors. Recent studies have shown that beta1-integrins co-localize with a variety of cytoskeletal complexes, signaling proteins and growth factor receptors. Since mechanosensitive ion channels and integrins have been proposed to participate in skeletal mechanotransduction, in this study, we investigated the possible co-localization of beta1-integrins with two ion channels and a P-type ATPase in mouse limb-bud chondrocytes. The alpha subunits of Na, K-ATPase, the epithelial sodium channel (ENaC) and the voltage activated calcium channel (VACC) were immunostained in organoid cultures derived from limb-buds of 12-day-old mice using well-characterized antibodies. Indirect immunofluorescence revealed abundant expression of beta1-integrins and each of the selected systems in limb-bud chondrocytes. Two-fluorochrome immunostaining demonstrated that beta1-integrin, Na, K-ATPase, ENaC and VACC co-localize in chondrocytes. Co-imunoprecipitation experiments revealed co-localization and association of integrins with ENaC, VACC and Na, K-ATPase. Cellular responses and signaling cascades initiated by the influx of calcium or sodium through putative mechanosensitive channels may be regulated more effectively if such channels were organized around integrins with receptors, kinases and cytoskeletal complexes clustered about them. The close proximity of ATPase ion pumps such as Na, K-ATPase to chondrocyte mechanoreceptor complexes could facilitate rapid homeostatic responses to the ionic perturbations brought about by activation of mechanically gated cation channels and efficiently regulate the intracellular milieu of chondrocytes.

Matrix changes during long-term cultivation of cartilage (organoid or high-density cultures).

In high density (organoid or micromass) cultures of prechondrogenic mesenchymal cells from limb buds of 12-day-old mouse embryos typical cartilaginous tissue develops after 3 days. Immunomorphological investigations have shown that it contains the typical components of the cartilaginous matrix, such as collagen type II and cartilage-specific proteoglycans. After a 2-week cultivation period hypertrophic cartilage cells develop to an increasing extent. Many of these cells as well as normal chondroblasts detach from the matrix from the 2nd week in vitro onwards to assume a fibroblast-like appearance. At the same time thick (25-65 nm) collagenous fibrils occur at the surface of these cells. These thick fibrils contain collagen type I, as shown by immunomorphology. Hence, in these older cartilage cultures chondroblasts change their synthesis programme or direction of differentiation. Consequently, a model for the study of "dedifferentiation" of cartilage and possibly also transformation of cartilage cells to osteoblasts has become available.

Integrins in ageing cartilage tissue in vitro.

Matrix-cell interactions are of great importance for numerous cell functions whereby integrins play an essential role as transmitters of extracellular signals. In cultures of ageing cartilage tissue (organoid or high density cultures) cartilage cells occur on the surface of which thick fibrils of collagen type I are deposited. Since integrins, in their role as receptors, cause an interaction between matrix components and cell membrane, we tried to demonstrate immunomorphologically (light and electron microscopically) the corresponding integrin receptors for collagen type I (beta 1 alpha 1 and beta 1 alpha 2) on the surface of these ageing cartilage cells. Cultures of normal, i.e. young cartilage tissue exhibit only beta 1 alpha 3- and beta 1 alpha 5-receptors; labelling against the integrins beta 1 alpha 1 and beta 1 alpha 2 is not possible in this case. Our results show that after the occurrence of thick fibrils cartilage cells express new receptors (beta 1 alpha 1 and beta 1 alpha 2) on the cell membrane. Thus, in ageing or dedifferentiating cartilage tissue it is not only the synthesis programme of matrix components (e.g. instead of collagen type II >> collagen type I) which changes but also the integrins (instead of alpha 3/beta 1, alpha 5/beta 1 >> alpha 1/beta 1, alpha 2/beta 1) so that new collagen types can be bound. These findings may also serve for a better understanding and interpretation of cartilage changes in vivo during ageing and under pathological conditions.

Co-localization of integrins and matrix metalloproteinases in the extracellular matrix of chondrocyte cultures.

Beta1-integrins were found in the cartilage matrix, suggesting their implication in the assembly of its architectural scaffold, but the mechanism for this event is not yet clear. Matrix metalloproteinases (MMPs) may be involved in an integrin-shedding mechanism and matrix beta1-integrins may act to alter MMP activity. To begin to address this question, this study was designed to determine whether beta1-integrins and MMPs are colocalized in the chondrocytes or in the extracellular matrix of cartilage. We investigated high-density cultures of limb buds of 12-day-old mouse embryos by double immunofluorescence, immunoelectron microscopy and by coimmunoprecipitation assays in order to examine the localization of beta1-integrins and matrix metalloproteinases (MMP-1, MMP-3 and MMP-9) in cartilage. It was found, that all investigated MMPs and beta1-integrins were specifically co-localized in high-density cartilage cultures. Immunogold and immunofluorescence labelling of both beta1-integrins and MMPs were observed not only at the surface of chondrocytes but mainly also in the pericellular space and distributed between collagen fibrils in the extracellular matrix (ECM) as well. Results of immunoprecipitation experiments suggest a functional association of MMPs and beta1-integrins in chondrocytes as already described for other cell types. Further investigations are needed to elucidate the functional association between beta1-integrins and MMPs in chondrocytes.

Effects of the antirheumatic remedy hox alpha--a new stinging nettle leaf extract--on matrix metalloproteinases in human chondrocytes in vitro.

Inflammatory joint diseases are characterized by enhanced extracellular matrix degradation which is predominantly mediated by cytokine-stimulated upregulation of matrix metalloproteinase (MMP) expression. Besides tumour necrosis factor-alpha (TNF-alpha), Interleukin-1beta (IL-1beta) produced by articular chondrocytes and synovial macrophages, is the most important cytokine stimulating MMP expression under inflammatory conditions. Blockade of these two cytokines and their downstream effectors are suitable molecular targets of antirheumatic therapy. Hox alpha is a novel stinging nettle (Urtica dioica/Urtica urens) leaf extract used for treatment of rheumatic diseases. The aim of the present study was to clarify the effects of Hox alpha and the monosubstance 13-HOTrE (13-Hydroxyoctadecatrienic acid) on the expression of matrix metalloproteinase-1, -3 and -9 proteins (MMP-1, -3, -9). Human chondrocytes were cultured on collagen type-II-coated petri dishes, exposed to IL-1beta and treated with or without Hox alpha and 13-HOTrE. A close analysis by immunofluorescence microscopy and western blot analysis showed that Hox alpha and 13-HOTrE significantly suppressed IL-1beta-induced expression of matrix metalloproteinase-1, -3 and -9 proteins on the chondrocytes in vitro. The potential of Hox alpha and 13-HOTrE to suppress the expression of matrix metalloproteinases may explain the clinical efficacy of stinging nettle leaf extracts in treatment of rheumatoid arthritis. These results suggest that the monosubstance 13-HOTrE is one of the more active antiinflammatory substances in Hox alpha and that Hox alpha may be a promising remedy for therapy of inflammatory joint diseases.

Interleukin-1beta-induced expression of the urokinase-type plasminogen activator receptor and its co-localization with MMPs in human articular chondrocytes.

The urokinase-type plasminogen activator receptor (uPAR) plays a critical role in cartilage degradation during osteoarthritis as it regulates pericellular proteolysis mediated by serine proteinases. Another important family of proteinases responsible for ECM destruction in arthritis are the matrix metalloproteinases (MMPs). MMPs are regulated by IL-1beta, a cytokine that plays a pivotal role in pathogenesis of osteoarthritis. This study was undertaken to address two questions: 1. Is uPAR-expression regulated by proinflammatory cytokines such as IL-1beta? 2. Does a functional co-localization exist between uPAR and MMPs? By immunohistochemical analysis we observed enhanced expression of uPAR on chondrocytes derived from osteoarthritic human cartilage compared to non-osteoarthritic controls. We found an IL-1beta-mediated expression of uPAR by immunoelectron microscopy. Western blot analysis demonstrated that IL-1beta-stimulated expression of uPAR on chondrocytes in vitro increased in a dose-dependent manner. Furthermore, we found a functional co-localization between uPAR and MMP-9 on IL-1beta-stimulated chondrocytes by means of a co-immunoprecipitation assay. Expression of uPAR in osteoarthritic cartilage but not in healthy cartilage suggests that uPAR plays a role in cartilage breakdown. We propose that uPAR-mediated effects e.g. pericellular proteolysis are one of other cytokine (IL-1beta)-mediated events that contribute to the pathogenesis of osteoarthritis. Furthermore, we found that MMPs and uPAR were part of the same cell surface complexes in chondrocytes. This finding underlines a functional interaction between MMPs and the serine proteinase system in the fine regulation of pericellular proteolysis. Interfering with uPAR signaling may present a novel target in arthritis therapy to prevent excessive proteolytic degradation.

Post-genomic applications of tissue microarrays: basic research, prognostic oncology, clinical genomics and drug discovery.

Tissue microarrays (TMAs) are an ordered array of tissue cores on a glass slide. They permit immunohistochemical analysis of numerous tissue sections under identical experimental conditions. The arrays can contain samples of every organ in the human body, or a wide variety of common tumors and obscure clinical cases alongside normal controls. The arrays can also contain pellets of cultured tumor cell lines. These arrays may be used like any histological section for immunohistochemistry and in situ hybridization to detect protein and gene expression. This new technology will allow investigators to analyze numerous biomarkers over essentially identical samples, develop novel prognostic markers and validate potential drug targets. The ability to combine TMA technology with DNA microarrays and proteomics makes it a very attractive tool for analysis of gene expression in clinically stratified tumor specimens and relate expression of each particular protein with clinical outcome. Public domain software allows researchers to examine digital images of individual histological specimens from TMAs, evaluate and score them and store the quantitative data in a relational database. TMA technology may be specifically applied to the profiling of proteins of interest in other pathophysiological conditions such as congestive heart failure, renal disease, hypertension, diabetes, cystic fibrosis and neurodegenerative disorders. This review is intended to summarize the strengths and weaknesses of TMA technology which will have an increasingly important role in the laboratories of the post-genomic era.

Synovial and peritoneal macrophages in organoid culture.

Cultivation of macrophages and their progenitors has been very useful for elucidation of function, behaviour and morphology of these cells. The purpose of this contribution is to describe a new in vitro system (organoid, high density or micromass culture) which proved to be convenient for cultivation of macrophages derived from human synovial fluid and tissue and mouse peritoneal fluid. Using this method, highly differentiated and functionally active macrophages of marked purity and long maintenance (up to 2 weeks) could be obtained even after previous cultivation and subcultivation in monolayer culture. The macrophages were identified by electron microscopy and immunomorphology using HLA-DR-DP, CD-68 (markers for human macrophages), anti-human-polymorphonuclear leukocyte-gelatinase and F4/80 (a mouse macrophage surface marker). The significance of this method as a research tool in the study of cartilage degradation by macrophages in co-cultures is stressed.

Glucose transport and metabolism in chondrocytes: a key to understanding chondrogenesis, skeletal development and cartilage degradation in osteoarthritis.

Despite the recognition that degenerative cartilage disorders like osteoarthritis (OA) and osteochondritis dissecans (OCD) may have nutritional abnormalities at the root of their pathogenesis, balanced dietary supplementation programs have played a secondary role in their management. This review emphasizes the importance and role of nutritional factors such as glucose and glucose-derived sugars (i.e. glucosamine sulfate and vitamin C) in the development, maintenance, repair, and remodeling of cartilage. Chondrocytes, the cells of cartilage, consume glucose as a primary substrate for ATP production in glycolysis and utilize glucosamine sulfate and other sulfated sugars as structural components for extracellular matrix synthesis and are dependent on hexose uptake and delivery to metabolic and biosynthetic pools. Data from several laboratories suggests that chondrocytes express multiple isoforms of the GLUT/SLC2A family of glucose/polyol transporters. These facilitative glucose transporter proteins are expressed in a tissue and cell-specific manner, exhibit distinct kinetic properties, and are developmentally regulated. They may also be regulated by endocrine factors like insulin and insulin-like growth factor I (IGF-I) and cytokines such as interleukin 1 beta (IL-1 beta) and tumour necrosis factor alpha (TNF-alpha). Recent studies suggest that degeneration of cartilage may be triggered by metabolic disorders of glucose balance and that OA occurs coincident with metabolic disease, endocrine dysfunction and diabetes mellitus. Based on these metabolic, endocrine and developmental considerations we present a novel hypothesis regarding the role of glucose transport and metabolism in cartilage physiology and pathophysiology and speculate that supplementation with sugar-derived vitamins and nutraceuticals may benefit patients with degenerative joint disorders.

Effects of ofloxacin on integrin expression on epiphyseal mouse chondrocytes in vitro.

Quinolone-induced arthropathy is an important toxic effect in immature animals that has led to restrictions of the therapeutic use of these antimicrobial agents. The effects of ofloxacin on epiphyseal chondrocytes from 17-day-old mouse foetuses were studied in vitro. Adhesion of the cells to culture dishes was impaired in a concentration-dependent manner and was first perceptible at a concentration of 10 mg ofloxacin/litre medium. A closer analysis by immunomorphological methods showed that the expression of several integrin receptors (beta1, alpha3, alpha5beta1, alphavbeta3) on the chondrocytes was reduced. Again, first alterations occurred at the rather low concentration of 10 mg ofloxacin/litre medium, and at 30 mg ofloxacin/litre medium alpha3- and alpha5beta1 integrins were demonstrable on 50% or less of the cultured cells. Based on these findings in vitro, a new hypothesis for the mechanism of the chondrotoxicity of quinolones is proposed: the ability of these antimicrobials to form chelate complexes with divalent cations could explain why the integrin receptors on chondrocytes are altered after quinolone exposure, since it is well known that the function of the integrin receptor depends on calcium or magnesium ions. Further investigations are under way to study the effects of quinolones on integrin receptors in cartilage in more detail.