Noise effects and filtering in controlled light exposure microscopy.

Phototoxicity and photobleaching are major limitations of fluorescence live-cell microscopy. A straightforward way to limit phototoxicity and photobleaching is reduction of the excitation light dose, but this causes loss of image quality. In confocal fluorescence microscopy, the field of view is illuminated uniformly whereas in controlled light exposure microscopy, illumination is controlled per pixel on the basis of two illumination strategies. The controlled light exposure microscopy foreground strategy discriminates between bright and weak foreground. Bright foreground pixels are illuminated with a reduced light dose resulting in limited excitation of fluorophores and consequently limited phototoxicity and photobleaching. The controlled light exposure microscopy background strategy discriminates between foreground and background. Pixels that are judged to be background are also illuminated with a reduced light dose. The latter illumination strategy may introduce artefacts due to the stochastic character of photon flow. These artefacts are visible as erratic 'darker pixels' in the foreground with a lower pixel value than the neighbouring pixels. This paper describes a special adaptive image processing filter that detects and corrects most of the 'darker pixels'. It opens the possibility to use controlled light exposure microscopy even in high noise (low signal to noise ratio) imaging to further reduce phototoxicity and photobleaching.

Mechanisms of heparin induced anti-cancer activity in experimental cancer models.

BACKGROUND: Retrospective analyses of clinical trials and prospective clinical studies have suggested that heparins may have an effect on cancer survival. This putative anti-cancer activity of heparins is supported by data from studies in animal tumour models. OBJECTIVE: To clarify the various potential mechanisms of heparin anti-cancer activity we evaluated the data from pre-clinical studies in which heparins have been tested as anti-cancer therapy. METHODS: Pre-clinical studies, published between 1960 and 2005 were assessed. Data were collected on the type and dose of heparin used, duration of exposure to heparin, interval between heparin administration and cancer cell inoculation, and the animal tumour model used. In addition, a distinction was made in the analysis between heparin effects on the primary tumour or on established metastases and effects on the metastatic potential of infused cells. RESULTS: Heparins seemed to affect the formation of metastasis rather than the growth of primary tumours. Chemically modified heparins with no or limited anticoagulant activity also showed anti-metastatic properties. Possible mechanisms to explain the effects on the process of metastases include inhibition of blood coagulation, inhibition of cancer cell-platelet and -endothelial interactions by selectin inhibition and inhibition of cell invasion and angiogenesis. CONCLUSION: The anti-cancer activity of heparins depends more on inhibition of metastasis formation than on the effects on primary tumour growth. These effects are probably related to both coagulation and non-coagulation dependent factors. For a definitive proof of the anti-cancer activity of heparins in the clinic, prospective randomized trials especially in patients with early metastatic disease or in the adjuvant setting are urgently needed.

Dietary omega-3 polyunsaturated fatty acids promote colon carcinoma metastasis in rat liver.

The effects of omega-3 polyunsaturated fatty acids (PUFAs) and omega-6 PUFAs on the development of experimentally induced colon carcinoma metastasis in rat liver were investigated quantitatively in vivo. Rats were kept on either a low-fat diet or on a fish oil (omega-3 PUFAs) or safflower oil (omega-6 PUFAs) diet for 3 weeks before the administration of colon cancer cells to the portal vein, until they were sacrificed at 1 or 3 weeks after tumor transplantation. At 1 week after transplantation, the fish oil diet had induced 7-fold more metastases (in terms of number and size) than had the low-fat diet, whereas the safflower oil diet had not affected the number and total volume of metastases. At 3 weeks after tumor transplantation, the fish oil diet and the safflower oil diet had induced, respectively, 10- and 4-fold more metastases (number) and over 1000- and 500-fold more metastases (size) than were found in the livers of rats on the low-fat diet. These differences were sex independent. Immunohistochemical analysis revealed that the immune system in the liver (Kupffer cells, pit cells, T cells, newly recruited macrophages, and the activation state of macrophages) did not play a significant role in this diet-dependent outgrowth of tumors. In conclusion, omega-3 and omega-6 PUFAs promote colon cancer metastasis in the liver without down-regulating the immune system. This finding has serious implications for the treatment of cancer patients with fish oil diet to fight cachexia.

The contribution of quantitative confocal laser scanning microscopy in cartilage research: chondrocyte insulin-like growth factor-1 receptors in health and pathology.

The use of immunohistochemical detection techniques and fluorescent molecular probes in light and fluorescence microscopy allows accurate and specific analysis of a great variety of cell and tissue components. However, when staining yields only low intensity levels, serious problems may arise with discrimination of specific signals against background staining. This problem is often inherent with articular cartilage research. Application of confocal laser scanning microscopy (CLSM) can circumvent these problems. The CLSM collects images that are almost free of out-of-focus signals, which results in improved spatial resolution and discrimination as compared with conventional microscopy. Moreover, CLSM allows optical sectioning of specimens and three-dimensional reconstruction of the microscopical object. Quantitative evaluation of microscopical images is hampered by out-of-focus signals because they interfere with specific signals in the image. Interference of these nonspecific signals can be diminished by application of CLSM; in CLSM only one single point in microscopical objects is illuminated at any time and this point is then imaged into the pinhole at the entrance of the photo-detector and subsequently digitized. The present review is a discussion of the present state of the art in digital imaging with the use of CLSM in cartilage research. This discussion includes aspects such as sensitivity, specificity, spatial resolution and accuracy of quantitative analysis in microscopical immunofluorescent objects.

Early effects of high doses of retinol (vitamin A) on the in situ cellular metabolism in rat liver.

Understanding of the possible toxicity associated with hypervitaminosis A becomes increasingly important in view of the popularity of vitamin A supplementation. Hypervitaminosis A for many years may eventually lead to hepatocellular damage. In the present study, rats were treated for 7 days with high doses of retinol to study the early effects on the metabolism of different types of liver cells using (enzyme) histochemistry, immunohistochemistry and electron microscopy. Excessive intake of vitamin A activates Kupffer cells and induces accumulation of lipid droplets in fat-storing cells as well as proliferation of these cells. Moreover, it affects the metabolic heterogeneity in the liver lobules, but does not lead to apparent cell damage. Based on the changes in marker enzymes for different metabolic processes, it is concluded that the capacity for breakdown of purines, the antioxidant capacity, the potential for phagocytosis and the regulation of ammonia levels were largely decreased. Increased alkaline phosphatase activity in hepatocytes pointed to an activated process of transport of retinol esters over the bile canalicular membrane. The possible causes of these metabolic changes have been described in the discussion.

Effects of n-3 and n-6 polyunsaturated fatty acid-enriched diets on lipid metabolism in periportal and pericentral compartments of female rat liver lobules and the consequences for cell proliferation after partial hepatectomy.

The effects of a low fat diet or diets enriched with either n-6 or n-3 polyunsaturated fatty acids (safflower or fish oil, respectively) on lipid metabolism in periportal and pericentral zones of female rat liver lobules were investigated in relation with cell proliferation after partial hepatectomy. It was found that cell proliferation was localized almost exclusively in periportal and midzonal areas and was significantly reduced by 60% after a fish oil diet only. The fish oil diet caused a strongly increased beta-oxidation capacity in peroxisomes and a moderately increased catalase activity. Catalase activity was mainly localized pericentrally, particularly after partial hepatectomy, whereas the capacity of lipid peroxidation product formation was doubled only in periportal zones in rats on a fish oil diet. The capacity of glucose-6-phosphate dehydrogenase activity to produce NADPH was distinctly lower in both zones of liver lobules as a result of the fish oil diet. Localization patterns and activity in liver lobules of NADPH-cytochrome c (P450) reductase were not significantly affected by fish oil diet. Therefore, it is concluded that elevated peroxisomal beta-oxidation and increased lipid peroxidation capacity in periportal zones of liver lobules coincide with reduced cell proliferation in hepatectomized rats on fish oil diet. These findings support the hypothesis that lipid peroxidation products are involved in the regulation of cell proliferation.

Cysteine proteinase activity in the development of arthritis in an adjuvant model of the rat.

Cathepsin B and L activity was studied histochemically in arthritic rat ankle joints using specific synthetic substrates in a post coupling method on unfixed and undecalcified cryostat sections of rat ankle joints. Activity was strongly increased in chondrocytes and cells of the inflamed synovium with the development of arthritis induced by the synthetic adjuvant CP20961. Activity reached a maximum 20 days after induction of arthritis and decreased as the rats entered natural remission. Cathepsin B and L were at their highest level when macrophages were present in the joint space, as shown by using monoclonal antibody markers for rat macrophages (ED1 and ED2) in a biotin-avidin immunoperoxidase assay. This suggests that the macrophage infiltrate may have stimulated proteinase production in chondrocytes through cytokine release. The profile of appearance of cysteine proteinases suggests their involvement in the breakdown of cartilage and bone in the arthritic joint.

Effects of dietary fatty acid composition on tumor growth and metastasis.

Unsaturated fatty acids of the n-6 and n-3 class have been shown to affect tumor growth and metastasis. The very long chain polyunsaturated fatty acids of the n-3 family, e.g. eicosapentaenoic acids (C20:5n-3) and docosahexaenoic acids (C22:5n-3), have an inhibiting effect on tumor growth. Metastasis is promoted by n-6 polyunsaturated fatty acids, e.g. linoleic acid (C18:2n-6) and gamma-linolenic acid (C18:3n-6). The mechanisms of promotion and inhibition are described in the present review. The mechanisms of lipid peroxidation, which appears to be an important factor in the inhibition of tumor growth, are discussed. Lipid peroxidation is induced by polyunsaturated fatty acids involving autoperoxidation a.o. and the enzymes cytochrome P450, cyclooxygenase and lipoxygenase. In tumor cells these enzymes are decreased in activity but at present the reason for this reduction is not known. Lipid peroxidation products such as hydroxyeicosatetraenoic acids (HETES), hydroperoxy eicosatetraenoic acids (HPETES) and malondialdehyde may have a regulating effect on DNA duplication enzymes (e.g. polymerases). Prostaglandin synthesis in tumor cells and macrophages is also affected by polyunsaturated fatty acids. The fish oil fatty acids are known to reduce prostaglandin synthesis by competing with arachidonic acid for the enzyme cyclooxygenase. However, fish oil fatty acids have an antagonistic effect on cyclooxygenase. Polyunsaturated fatty acids also have an effect on the immune system and particularly on macrophages. Macrophages, but also T-cells and B-cells, are inhibited by prostaglandins such as PGE2, while immunosuppressor cells are stimulated by PGE2.

The effects of interleukin-1 on articular cartilage destruction as observed in arthritic diseases, and its therapeutic control.

At present there is substantial evidence to suggest that interleukin 1 (IL-1) may act as a key mediator in the normal physiologic regulation of cartilage as well as in the pathogenesis of cartilage destruction in arthritic disorders. IL-1 induces stimulation of chondrocyte catabolism and alters chondrocyte biosynthesis in articular cartilage. These actions of IL-1 may lead to destruction and inappropriate repair following degradation of the cartilage matrix. Moreover, IL-1 induced biological activities in chondrocytes may be influenced by growth factors (e.g. fibroblast growth factor, insulin-like growth factor, transforming growth factor-beta), guanine nucleotide proteins, or other cytokines. With respect to the widely suggested potential significance of IL-1 in arthritis, pharmacological control of IL-1 action is of important clinical relevance. Today the therapeutic control of IL-1 induced effects in articular cartilage destruction as observed in arthritic diseases can be divided into drugs which affect IL-1 production, drugs which modify or block the IL-1 effect before stimulation of the target cell, or drugs that interfere with the IL-1 induced effects, e.g. steroidal drugs, non-steroidal anti-inflammatory drugs, immunoregulatory drugs or class-specific proteinase inhibitors. However, these drugs do not specifically block IL-1 activity. For the development of therapeutic agents capable of specifically blocking IL-1 effects, a better understanding of IL-1 induced activities is needed. In conclusion, knowledge about chondrocyte metabolic and regulatory alterations would be beneficial in unraveling the events that take place in arthritic diseases and would favor therapeutic research for agents that might arrest the progressive destruction of articular cartilage in pathological conditions.