Transendothelial migration of peripheral blood mononuclear cells (PBMCs) and their subsequent interaction with the subendothelial matrix lead to their differentiation to macrophages (mphis). To study whether preexposure of monocytes in circulation to modified proteins influences their differentiation to mphis, an in vitro model system using isolated PBMC in culture was used. The effect of modified proteins such as oxidatively modified LDL (ox-LDL), acetylated and non-enzymatically glycated-BSA (NEG-BSA) on the differentiation process was studied by monitoring the upregulation of mphi specific functions such as endocytosis, production of matrix metalloproteinases (MMPs), expression of surface antigen, activity of beta-glucuronidase and down regulation of monocyte specific myeloperoxidase activity. Rate of endocytosis, production of MMPs and beta-glucuronidase activity were significantly greater in cells treated with modified proteins irrespective of the nature of modification. Both CuSO4 ox-LDL and HOCl ox-LDL increased the rate of expression of the mphi specific functions. FACS analysis showed that the rate of upregulation of mphi specific CD71 and down regulation of monocyte specific CD14 were high in cells supplemented with modified proteins. Studies using PPARgamma antagonist and agonist suggest its involvement in CuSO4 ox-LDL induced monocyte-macrophage (mo-mphi) differentiation whereas the expression of macrophage specific functions in cells exposed to other modified proteins was independent of PPARgamma. PBMC isolated from hypercholesterolemic rabbits in culture expressed mphi specific functions at a faster rate compared to normal controls indicating that these observations are relevant in vivo. These results indicate that preexposure of monocytes to modified proteins promote their differentiation to mphis and may serve as a feed forward type control for clearing modified proteins.

Transendothelial migration of monocytes followed by their differentiation into macrophages involves interaction of monocytes with subendothelial matrix. The influence of extracellular matrix on monocyte-macrophage differentiation was studied using an in vitro model system with human PBMC maintained on different matrix protein substrata. Upregulation of macrophage specific marker activities such as endocytosis of modified proteins, changes in expression of cell surface antigen, and production of matrix metalloproteinases was studied. Cells maintained on Fibronectin (Fn) showed significantly higher rate of endocytosis and production of MMP2 and MMP9 when compared to other matrix protein substrata. Immunoblot analysis, ELISA, and zymography showed that Fn-dependent upregulation of MMPs was blocked by antibodies to alpha(5)beta(1) integrin indicating that the Fn effect was mediated by integrins. The Fn effect on mo-mPhi was blocked by genistein and herbimycin. As monocytes differentiate to macrophages there was an increase in the rate of production of Fn. These results indicate the influence of the microenvironment of the cell, particularly Fn, on mo-mPhi differentiation and integrin-mediated downstream signaling through focal adhesion kinase and Src type tyrosine kinase is involved in this.

Formation of foam cells from macrophages, which are formed by the differentiation of blood-borne monocytes, is a critical early event in atherogenesis. To examine how pre-exposure of monocytes to modified proteins, such as oxLDL, influences their differentiation to macrophages, an in vitro model system using isolated PBMC maintained in culture in the presence of oxLDL was used. Pretreatment of monocytes with oxLDL caused a faster rate of expression of macrophage-specific functions and loss of monocyte-specific functions compared to unmodified LDL. The effect of oxidation of lipid component of LDL by CuSO(4) and its protein component by HOCl, on mo-mϕ differentiation was studied by monitoring the upregulation of macrophage-specific functions, particularly MMP-9. Chloroquine, a lysosomal degradation blocker, significantly reversed the effect mediated by CuSO(4) oxLDL, indicating the involvement of lysosomal degradation products, while no such effect was observed in HOCl oxLDL-treated cells, indicating the existence of a pathway independent of its lysosomal degradation products. Reversal of the effect of oxLDL by NAC and Calphostin C, an inhibitor of PKC, suggested the activation of RO-mediated signaling pathways. Use of inhibitors of signaling pathways showed that CuSO(4) oxLDL upregulated mϕ-specific MMP-9 through p38 MAPK and Akt-dependent pathways, while HOCl oxLDL utilized ERK ½ and Akt. Further analysis showed the activation of PPARγ and AP-1 in CuSO(4) oxLDL, while HOCl-oxLDL-mediated effect involved NFκB and AP-1. These results suggest that lipid oxLDL- and protein oxLDL-mediated upregulation of mo-mϕ-specific functions involve lysosomal degradation-dependent and -independent activation of intracellular signaling pathways.

Photodynamic therapy (PDT) involves the use of photochemical reactions mediated through the interaction of photosensitizing agent for the treatment of malignant tumor. The present study was carried out to evaluate the photosensitizing potential of embelin isolated from Embelia ribes. For PDT study, cells were grouped into four groups. Two types of control were used, one with Ehrlich's ascites carcinoma (EAC) cells alone (group 1) and one which received the illumination alone (group 2). Group 3 contains cells treated with different concentrations of embelin for 1 h at 37 °C. Another set of cells (group 4) after treatment with different concentrations of embelin was illuminated in ice for 4 min with a 1000-W halogen lamp. To study the mechanism of cell death, the following parameters such as reactive oxygen species (ROS), caspase-3, lactate dehydrogenase (LDH), and thiobarbituric acid reactive substances (TBARS) were analyzed both in the absence and presence of light after the treatment of embelin. PDT study clearly indicated that embelin alone recorded no cytotoxicity, but for light treatment alone with the different concentrations of embelin, there was a significant induction of cytotoxicity in a concentration-dependent manner. Level of ROS and LDH increased in cells treated with embelin. Moreover, caspase-3 also increased which clearly indicated that cell death is through caspase-dependent apoptosis. This is the first report on PDT, a novel modality, using embelin for cancer therapy in vitro. The novelty of the study depends on the fact that cytotoxicity was produced by the synergistic effect of the embelin and light.

Background: Disulfiram was widely used as an aversive agent for treatment of alcohol dependence since its discovery. Various adverse drug reactions have been documented. Psychosis due to disulfiram is not a common side effect. Methods & Discussion: We report two cases of disulfiram associated psychosis. We also highlight the biological mechanism of the psychosis associated with disulfiram use. The Dopamine (DA) hypothesis of schizophrenia states that an increase in DA activity in certain brain areas is associated with psychotic symptoms in schizophrenic patients. The possible explanation for the development of psychosis, might be inhibition of Dopamine-Betahydroxylase (DBH) enzyme due to metabolites of disulfiram. This enzyme catalyzes the metabolism of DA into norepinephrine. Inhibition of DBH increases dopamine level. Furthermore, alcoholics who developed psychosis due to disulfiram were found to have low levels of amine and monoamine oxidase, suggesting DBH blockage. Conclusion: Disulfiram induced psychosis is not an uncommon side effect. Biological mechanism of Disulfiram induced psychosis, probably via its DBH inhibiting effects, which lead to an increase in DA activity. Hence, careful monitoring is required for emergent of psychotic symptoms in vulnerable patients while on disulfiram treatment.