In this study, the human chondrosarcoma cell line SW1353 was investigated by gene expression analysis in order to validate it as an
in vitro model for primary human (adult articular) chondrocytes ...(PHCs).
PHCs and SW1353 cells were cultured as high density monolayer cultures with and without 1
ng/ml interleukin-1β (IL-1β). RNA was isolated and assayed using a custom-made oligonucleotide microarray representing 312 chondrocyte-relevant genes. The expression levels of selected genes were confirmed by real-time polymerase chain reaction and the gene expression profiles of the two cell types, both with and without IL-1β treatment, were compared.
Overall, gene expression profiling showed only very limited similarities between SW1353 cells and PHCs at the transcriptional level. Similarities were predominantly seen with respect to catabolic effects after IL-1β treatment. In both cell systems matrix metalloproteinase-1 (MMP-1), MMP-3 and MMP-13 were strongly induced by IL-1β, without significant induction of MMP-2. IL-6 was also found to be up-regulated by IL-1β in both cellular models. On the other hand, intercellular mediators such as leucemia inhibitory factor (LIF) and bone morphogenetic protein-2 (BMP-2) were not induced by IL-1β in SW1353 cells, but significantly up-regulated in PHCs. Bioinformatical analysis identified nuclear factor kappa-B (NFκB) as a common transcriptional regulator of IL-1β induced genes in both SW1353 cells and PHCs, whereas other transcription factors were only found to be relevant for individual cell systems.
Our data characterize SW1353 cells as a cell line with only a very limited potential to mimic PHCs, though SW1353 cells can be of value to study the induction of protease expression within cells, a phenomenon also seen in chondrocytes.
Abstract Objectives Angiogenesis is essential for tumor growth and metastasis. An association between microvessel density, a measure of tumor angiogenesis, and conventional prognostic variables has ...been shown for many different tumor entities. In extrahepatic cholangiocarcinoma, the VEGF expression and microvessel density have rarely been investigated. Methods Paraffin-embedded specimens from 51 resected adenocarcinomas of the extrahepatic bile duct were immunostained for vascular endothelial growth factor A (VEGF A) and CD 34 to evaluate the microvessel density (MVD). VEGF A staining was evaluated by combining intensity and percentage of positive tumor cells, as low (expression equal or below the median), or high (above the median). Microvessel density was assessed using a method published by Weidner et al. Results Median disease free survival (DFS) of the study group was 12.5 months (range, 1–66.3 months). DFS was calculated in the 39 patients with complete resection. It was significantly better in patients with low microvessel density than DFS in patients with high microvessel density (33 months (range, 3–66.3 months) vs. 21.8 months (range, 1.6–31.6 months); p = 0.022). In contrast, VEGF A expression did not correlate with survival. There was a trend toward a higher VEGF A expression in highly vascularized tumors ( p = 0.08), but failed to reach statistic significance. Conclusions The present study indicates, that vascularisation has an important impact on survival of extrahepatic cholangiocarcinoma patients. Other molecules than VEGF A are probably involved in neovascularization in extrahepatic cholangiocarcinoma.
1. The results of neuropsychological, neuropharmacological, and neurophysiological experiments have implicated the ventral striatum in reward-related processes. We designed a task to allow us to ...separate the effects of sensory, motor, and internal signals so that we could study the correlation between the activity of neurons in the ventral striatum and different motivational states. In this task, a visual stimulus was used to cue the monkeys as to their progress toward earning a reward. The monkeys performed more quickly and with fewer mistakes in the rewarded trials. After analyzing the behavioral results from three monkeys, we recorded from 143 neurons from two of the monkeys while they performed the task with either juice or cocaine reward. 2. In this task the monkey was required to release its grip on a bar when a small visual response cue changed colors from red (the wait signal) to green (the go signal). The duration of the wait signal was varied randomly. The cue became blue whenever the monkey successfully responded to the go signal within 1 s of its appearance. A reward was delivered after the monkey successfully completed one, two, or three trials. The schedules were randomly interleaved. A second visual stimulus that progressively brightened or dimmed signaled to the monkeys their progress toward earning a reward. This discriminative cue allowed the monkeys to judge the proportion of work remaining in the current ratio schedule of reinforcement. Data were collected from three monkeys while they performed this task. 3. The average reaction times became faster and error rates declined as the monkeys progressed toward completing the current schedule of reinforcement and thereby earning a reward, whereas the modal reaction time did not change. As the duration of the wait period before the go signal increased, the monkeys reacted more quickly but their error rates scarcely changed. From these results we infer that the effects of motivation and motor readiness in this task are generated by separate mechanisms rather than by a single mechanism subserving generalized arousal. 4. The activity of 138 ventral striatal neurons was sampled in two monkeys while they performed the task to earn juice reward. We saw tonic changes in activity throughout the trials, and we saw phasic activity following the reward. The activity of these neurons was markedly different during juice-rewarded trials than during correctly performed trials when no reward was forthcoming (or expected). The responses also were weakly, but significantly, related to the proximity of the reward in the schedules requiring more than one trial. 5. The monkeys worked to obtain intravenous cocaine while we recorded 62 neurons. For 57 of the neurons, we recorded activity while the monkeys worked in blocks of trials during which they self-administered cocaine after blocks during which they worked for juice. Although fewer neurons responded to cocaine than to juice reward (19 vs. 33%), this difference was not significant. The neuronal response properties to cocaine and juice rewards were independent; that is, the responses when one was the reward one failed to predict the response when the other was the reward. In addition, the neuronal activity lost most of its selectivity for rewarded trials, i.e, the activity did not distinguish nearly as well between cocaine and sham rewards as between juice and sham rewards. 6. Our results show that mechanisms by which cocaine acts do not appear to be the same as the ones activated when the monkeys were presented with an oral juice reward. This finding raises the intriguing possibility that the effects of cocaine could be reduced selectively without blocking the effects of many natural rewards.
Single neurons in the ventral striatum of primates carry signals that are related to reward and motivation. When monkeys performed a task requiring one to three bar release trials to be completed ...successfully before a reward was given, they seemed more motivated as the rewarded trials approached; they responded more quickly and accurately. When the monkeys were cued as to the progress of the schedule, 89 out of 150 ventral striatal neurons responded in at least one part of the task: (1) at the onset of the visual cue, (2) near the time of bar release, and/or (3) near the time of reward delivery. When the cue signaled progress through the schedule, the neuronal activity was related to the progress through the schedule. For example, one large group of these neurons responded in the first trial of every schedule, another large group responded in trials other than the first of a schedule, and a third large group responded in the first trial of schedules longer than one. Thus, these neurons coded the state of the cue, i.e., the neurons carried the information about how the monkey was progressing through the task. The differential activity disappeared on the first trial after randomizing the relation of the cue to the schedule. Considering the anatomical loop structure that includes ventral striatum and prefrontal cortex, we suggest that the ventral striatum might be part of a circuit that supports keeping track of progress through learned behavioral sequences that, when successfully completed, lead to reward.
Gene expression analysis including large scale gene expression profiling has become a very basic tool for investigating the pathogenesis of degenerative joint diseases as well as for the search of ...new drug targets. However, gene expression analysis so far revealed very complex expression patterns rather than a clear picture of molecular changes occurring during the initiation and progression of the disease. To elucidate the molecular changes in osteoarthritis the analysis of the fetal growth plate as a developmental model for phenotypic changes in chondrocytes occurring in osteoarthritis can help in three ways: it allows to interpret gene expression patterns in the context of disease-relevant processes also occurring in developing cartilage (e.g. cell differentiation, proliferation, matrix synthesis, catabolism and calcification), it offers the chance to investigate gene function in these functional contexts by knocking out or overexpressing genes in animals, and it provides a suitable model for testing the effect of therapeutic compounds on these processes within the growing cartilage.
Bone morphogenetic protein-7 (BMP)-7 plays an important role during fetal kidney development. In the adult, BMP-7 is most strongly expressed in the kidney compared to other organs, but the exact ...expression pattern as well as the function of BMP-7 is unclear. The major aim of the present study was to define which parts of the human kidney do physiologically express BMP-7 and which cells appear to be targets of BMP activity by showing phosphorylated BMP-receptor-associated Smads 1, 5, or 8 and inhibitor of differentiation factor 1 (ID1) expression. BMP-7 expression was localized by immunohistology to the epithelia of the distal tubule as well as the collecting ducts (CDs). Phospho-Smads 1/5/8 and ID1 expression largely colocalized with BMP-7 and was also localized in the epithelia of the distal tubule and the CDs. This was confirmed by polymerase chain reaction-based mRNA expression analysis. In vitro, proximal tubular cells (PTCs) expressed BMP receptors and BMP-receptor-associated Smads and were reactive to BMP-7. Our data indicate that BMP-7 expression in the adult human kidney appears to be more restricted than in the fetal situation and predominantly found in the distal nephron. Also, evidence of in vivo BMP signalling (i.e. phospho-Smads and ID1 expression) was found there. These findings suggest that BMP-7 plays a physiological role mostly in this part of the kidney. Still, as reported previously, PTCs are responsive to BMP-7, but presumably not in an autocrine or paracrine mode in normal adult kidneys.
Summary Objective The development of a reliable high-throughput transfection protocol for primary human articular chondrocytes. Methods Primary human chondrocytes were isolated from adult knee ...cartilage by an optimized enzymatic digestion protocol and cultivated in high-density monolayer culture for 3–5 days. Isolated chondrocytes were transfected with a green fluorescent protein (GFP)-expressing reporter construct using amaxa's Nucleofector 96-well Shuttle® System. Transfection efficiencies were measured by fluorescence activated cell sorting and cell viability was determined by an adenosine-5′-triphosphate (ATP) assay. siRNA oligonucleotides (against glyceraldehyde-3-phosphate dehydrogenase (GAPDH)) were transfected into the cells using the optimized nucleofection protocol and mRNA knockdown values were determined by a branched-DNA assay. Results Transfection efficiencies of more than 70% of surviving cells were achieved routinely with the nucleofection protocol presented in this article. Cell viability 24 h post transfection was around 80%. The cell number used per transfection was reduced to 2 × 105 per sample. In addition, the protocol proved to be well suited for the transfer of siRNA molecules into primary human chondrocytes with suppression rates on the mRNA level of more than 95% (for GAPDH). Conclusions We present the successful use of nucleofection on primary human chondrocytes using a microtiter plate compatible format that for the first time allows the efficient transfection of up to 96 samples in parallel. The optimized nucleofection protocol is offering maximum substrate flexibility by allowing transfer of DNA and siRNA oligonucleotides with the same set of parameters. Moreover, the transfection procedure requires substantially lower cell numbers than single cuvette protocols and is therefore perfectly suited for applications requiring multiple experimental replicates.
Objective: To investigate quantitatively the mRNA expression levels of YKL-40, an established marker of rheumatoid and osteoarthritic cartilage degeneration in synovial fluid and serum, and a closely ...related molecule YKL-39, in articular chondrocytes. Methods: cDNA array and online quantitative polymerase chain reaction (PCR) were used to measure mRNA expression levels of YKL-39 and YKL-40 in chondrocytes in normal, early degenerative, and late stage osteoarthritic cartilage samples. Results: Expression analysis showed high levels of both proteins in normal articular chondrocytes, with lower levels of YKL-39 than YKL-40. Whereas YKL-40 was significantly down regulated in late stage osteoarthritic chondrocytes, YKL-39 was significantly up regulated. In vitro both YKLs were down regulated by interleukin 1β. Conclusions: The up regulation of YKL-39 in osteoarthritic cartilage suggests that YKL-39 may be a more accurate marker of chondrocyte activation than YKL-40, although it has yet to be established as a suitable marker in synovial fluid and serum. The decreased expression of YKL-40 by osteoarthritic chondrocytes is surprising as increased levels have been reported in rheumatoid and osteoarthritic synovial fluid, where it may derive from activated synovial cells or osteophytic tissue or by increased matrix destruction in the osteoarthritic joint. YKL-39 and YKL-40 are potentially interesting marker molecules for arthritic joint disease because they are abundantly expressed by both normal and osteoarthritic chondrocytes.
Subtyping of osteoarthritic synoviopathy Oehler, S; Neureiter, D; Meyer-Scholten, C ...
Clinical and experimental rheumatology,
09/2002, Letnik:
20, Številka:
5
Journal Article
Recenzirano
Osteoarthritis research is traditionally concentrating on events within the degenerated articular cartilage. Changes in the synovial membrane are largely neglected. In fact, they are generally ...interpreted as secondary to the cartilage changes and not pathogenetically involved in the disease process. In this study, we present a systematic analysis of the synovial reaction pattern in early and late stages of the osteoarthritic disease process.
A large series of synovial specimens derived from early and late stage osteoarthritic cartilage disease were investigated by histological and immunohistochemical means for tissue architecture and inflammatory cell infiltrates. For comparison, also samples with rheumatoid arthritis, seronegative arthritis, and septic arthritis were included as well as normal synovial membrane specimens.
In all specimens derived from patients with diagnosed osteoarthritis alterations of the synovial tissue were observed. A large spectrum of alterations was found in different stages of osteoarthritic joint disease and four different basic pattern of synovial reactions could be identified: (i) hyperplastic, (ii) inflammatory, (iii) fibrotic, and (iv) detritus-rich synoviopathy.
We show that in all cases of clinically overt osteoarthritic joint disease significant synovial pathology is associated. Furthermore, our study clearly documents that in osteoarthritic synovium significant inflammation can occur. This is suggestive of a distinct pathogenetic role of the synovium also in osteoarthritic cartilage degeneration at least in a subset of cases.
Osteoarthritis, the degeneration of the joints, is the leading source of physical disability with severely impaired quality of life due to pain and loss of joint functioning in industrialized ...nations. Clinically, degeneration affects mostly the large weight bearing joints of the legs like the hip or the knees, but in principle it can affect any joint of the body. Osteoarthritis represents a disease group with disease subsets that have different underlying pathophysiological mechanisms. Therefore primary osteoarthritis has to be distinguished from secondary forms of the disease, which are due to traumatic events, endocrine or metabolic disorders etc. The enormous frequency of this disease makes osteoarthritis one of the most expensive conditions in the Western world, both in terms of direct as well as indirect costs. So far, despite intensive efforts over several decades, the success of disease-modifying approaches have been rather limited and mostly restricted to analgesis and non-pharmacologic therapy (e.g. nonsteroidal anti-inflammatory agents, exercise, and physiotherapy). Joint replacement is still the unsurpassed therapy for the symptomatic relief of advanced and incapacitating OA. It is evident that there is a great need for the development of disease modifying agents in order to improve quality of life as well as to relieve the community of the enormous socio-economic burden of the disease.