• Advances in Traditional Medicine
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• v.5 no.3
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• pp.216-230
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• 2005

Although the field of study in immune enhancing compounds is relatively new, natural products from plants represent a rich and promising source of novel molecules with immunomodulating properties, Microglial cells, the main immune effector cells of the brain, usually display a ramified morphology and low expression levels of immunologically relevant antigens such as MHC class I and class II. Since any compound which participates in activation of phagocytic cells contributes to the production of potentially toxic factors, the search for convenient in vitro test-systems and study of mechanisms of action of these agents are of great interest. Human blood polymorphonuclear (PMN) cells and primary microglial cells isolated from Sprague-Dawley rats were used as cellular screening tests for study of phagocytosis-stimulating action of immunomodulating agents. Numbers of phagocytic activity were evaluated by the phagocyte ingestion of yeast cells and NO-synthase activity, nitrite production, and nitroblue tetrazolium test were determined after phagocyte stimulation. It was possible to demonstrate that indexes of phagocytic activity can be used as quantitative indicators for measurement immunomodulating activity. As a positive control, Zymosan A-induced phagocytosis in both PMN cells and primary microglial cells was used. $IFN-{\gamma}$ (0.1 -1 U/ml) stimulated phagocytosis in PMN cells 1.2 times after 2 - 3 h incubation, although at higher concentrations (10 - 100 U/ml) it strongly inhibited phagocytosis. In a similar way, at higher concentrations, $IFN-{\gamma}$ (100 - 500 U/ml) suppressed phagocytosis in zymosan-A stimulated microglial cells. When Polypodium leucotomus, cambricum and vulgare extracts were tested alone, increased levels of phagocytosis were observed in PMN. In addition, microglial cells showed both increased phagocytosis and MHC class-II antigen expressions. Surprisingly, when PMN and microglia were treated with a combination of Polypodium and $IFN-{\gamma}$, phagocytosis was not inhibited. We did not find changes in NO-synthase activity and nitrite production in both microglia and PMN cells activated by different immunomodulating agents. These results indicate that primary microglial cell cultures as well as human PMN cells can provide reproducible quantitative results in screening phagocytic activity of different immunoactive compounds. Furthermore, both inhibitory or activation mechanisms might be studied using these in vitro experimental approaches.

• Applied Microscopy
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• v.40 no.2
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• pp.73-80
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• 2010

Liver regeneration is a result of highly coordinated proliferation of hepatocytes and nonparenchymal liver cells. Partial hepatectomy (PH) is the most often used stimulus to study liver regeneration because, compared with other methods that use hepatic toxins, it is not associated with the tissue injury and inflammation, and the initiation of the regenerative stimulus is precisely defined. Granulocyte macrophage-colony stimulating factor (GM-CSF), which is a cytokine able to regulate the proliferation and differentiation of epithelial cells, was first identified as the most potent mitogen for bone marrow. Particularly, rrhGM-CSF, which is highly glycosylated and sustained longer than any other types of GM-CSF in the blood circulation, was specifically produced from rice cell culture. In this experiment, effects of rrhGM-CSF administration were evaluated in the regenerating liver after 78% PH of rats. Morphological changes induced by PH were characterized by destroyed hepatocyte plate around the central vein and enlarged nuclear cytoplasmic ratio and increased hepatocytes with two nuclei. And then, proliferation of liver cells (parenchymal and nonparenchymal) and rearrangement of plates and lobules seemed to be carried out during liver regeneration. These alterations in the experimental group preceded those of the control. Since proliferating cell nuclear antigen (PCNA) is known to be a nuclear protein maximally elevated in the S phase of proliferating cells, the protein was used as a marker of liver regeneration after PH in rats. PCNA levels by western blot analysis and immunohistology were compared between the two groups. PCNA protein expression of two groups at 12 hr and 24 hr after injury showed similar pattern. The protein expression showed the peak at 3 days in both groups, however, the protein level of the experimental group was higher than that of the control. On immunohistochemical observations, the reaction product of PCNA was localized at the nuclei of proliferating cells and the positive reaction in experimental group at 3 days was clearly stronger than that in control group. The results by Western blotting and immunohistology for PCNA showed similar pattern in terms of the protein levels. In conclusion, rrhGM-CSF administration during liver regeneration after 78% PH accelerated breakdown and restoration of the hepatic plate and expression of PCNA. These results suggest that rrhGM-CSF might play an important role during liver regeneration in rats.