Medical electron microscopy : official journal of the Clinical Electron Microscopy Society of Japan最新文献

We investigated the effectiveness of in vivo electrogene transfer as a means of therapy in rat urinary bladder carcinoma and in mammary carcinoma models in both athymic and syngeneic mice using the herpes simplex virus 1 thymidine kinase (HSVtk) or IL-12 genes in combination with ganciclovir (GCV). A significant increase in the levels of tissue apoptosis and necrosis was induced with a single injection of HSVtk vector directly into bladder and mammary tumors followed by in vivo transfection and a regimen of intraperitoneal GCV injection. This procedure induced significant selective tumor cell death, characterized by marked inflammation and peripheral macrophage influx. Active caspase-3 was also strongly expressed in areas of cell death, indicating the initiation of apoptosis. This result was confirmed in corollary in vitro studies on a mouse bladder carcinoma cell line in which elevated caspase-3, -8, and -9 activities and decreased mitochondrial membrane potential were observed as a result of transfection with HSVtk and addition of GCV to the medium. In the syngeneic mouse mammary cancer model, we additionally found both tumor volume and metastasis to lymph nodes and lungs to be significantly reduced throughout the 2-month experiment. However, in contrast to their syngeneic counterparts, HSVtk/GCV therapy did not effectively inhibit mammary tumor growth/metastasis in an athymic mouse model, leading us to believe that T-cell-mediated immune responses may participate via the bystander effect in HSVtk/GCV experimental therapy. We subsequently evaluated the antitumor activity of IL-12, which can activate T-cell-mediated immune responses involving macrophages, in the syngeneic mammary tumors and found that IL-12 also significantly suppressed mammary tumor growth and metastasis. We thus suggest that in vivo electrogene transfer is a useful transfection tool in cancer gene therapy and, in addition, we show that T-cell-mediated immune responses may be a critical factor in cancer gene therapy using HSVtk/GCV and IL-12.

Fukuyama-type congenital muscular dystrophy (FCMD), muscle-eye-brain disease (MEB), and Walker-Warburg syndrome (WWS) are congenital muscular dystrophies associated with central nervous system (CNS) lesions, represented by cobblestone lissencephaly and eye anomalies. The glia limitans, formed by astrocytic endfeet and covered with the basement membrane, is disrupted in fetal cases of these diseases. A gene responsible for FCMD is fukutin and that for MEB is protein O-linked mannose beta1,2-N-acetylglucosaminyltransferase (POMGnT1). Mutations in protein-O-mannosyltransferase 1 (POMT1) have been found in some WWS cases. POMGnT1 and POMT1 are involved in glycosylation of alpha-dystroglycan, which is one of the components of dystrophin-glycoprotein complex, linking dystrophin and extracellular matrix proteins at the basement membrane. Fukutin seems to have similar functions to those of POMGnT1 and POMT1, but its functions still remain to be clarified. In situ hybridization reveals that fukutin, POMGnT1, and POMT1 are expressed especially in astrocytes. Decrease of glycosylated alpha-dystroglycan has been reported in the skeletal muscle of FCMD, MEB, and WWS. Moreover, decrease of fukutin and glycosylated alpha-dystroglycan is observed in the brain of FCMD cases. Because astrocytes are involved in basement membrane formation at the glia limitans, fukutin, POMGnT1, and POMT1 are considered to relate to the pathogenesis of CNS lesions, and fukutin may be related to glycosylation of alpha-dystroglycan. Fukutin, POMGnT1, and POMT1 are expressed in immature neurons, suggesting they are also involved in neuronal migration itself. POMGnT1 and POMT1 are expressed in many mature neurons, but fukutin is positive in a few mature neurons. FCMD is a rather mild disease among FCMD, MEB, and WWS, and POMGnT1 and POMT1 seems to have more critical roles compared to fukutin in mature neurons.

We have proposed in the past that chest wall fibroblasts are transformed to regenerated mesothelial cells. This study was conducted to investigate the effects of prednisolone on the differentiation and migration of fibroblasts in their transformation to mesothelial cells. Rat fibroblasts harvested from intercostal thoracic wall specimens were cultured in culture medium until cell spheroids were formed. An experimental cell spheroid group to whose culture medium prednisolone had been added and a control spheroid group with no addition of prednisolone were then subjected to immunohistochemical and ultrastructural studies of the changes in the fibroblasts with the passage of time. On days 1 and 2 of culture, the fibroblasts in each group were cytokeratin negative. However, on day 3 the control group became cytokeratin positive, and ultrastructural observations revealed formation of macula adherens and microvilli. In contrast, the experimental group fibroblasts remained cytokeratin negative even on day 3, but became cytokeratin positive on day 5 of culture. Macula adherens and microvilli also manifested on day 5. Prednisolone inhibited the differentiation and migration of fibroblasts, but it was surmised that fibroblasts that have resisted from the effects of prednisolone finally differentiate into mesothelial cells which have formed macula adherens.

Growing evidence revealed that liver sinusoidal endothelial cells (SEC) play several important roles in physiology and pathology of the liver. It has been well understood that their structural characteristics, such as the membrane sieve and lack of basement membrane, facilitate direct contact of soluble and insoluble serum substances with hepatic parenchymal cells, resulting in enhancement of hepatic metabolic activity. In addition, SEC is now regarded as a member of the scavenger endothelial cells, which have potential to eliminate a variety of macromolecules from the blood circulation by receptor-mediated endocytosis. It is reported that molecules preferentially eliminated by SEC are denatured or modified proteins such as advanced glycation end products, extracellular matrix components including hyaluronic acid, and some lipoproteins. The nature of the scavenger receptors corresponding to these molecules remains to be clarified. Recently, it was noted that SEC has an antigen-presenting function similar to dendritic cells. Taken together, it is suggested that SEC, cooperating with Kupffer cells and hepatic dendritic cells, may partake of immunoregulatory functions in the liver. SEC also plays a pivotal role in the pathological process of ischemia-reperfusion injury following liver surgery and liver transplantation. Thus, it is of importance to elucidate the mechanisms of apoptosis and proliferation of SEC. Recent results on the regulation of growth and apoptotic signaling of SEC are discussed.

The presence of microfilaments in the vicinity of sinusoidal endothelial fenestrae (SEF) suggests that the cytoskeleton of liver sinusoidal endothelial cells (LSEC) plays an important role in the modulation of SEF. In this study, we investigated actin filaments around SEF in LSECs. Monolayers of LSEC culture were established by infusing a rat liver with collagenase for 30 min and then culturing in RMPI medium for 24 h. Cells were reacted with 0.1% Triton X for 5 s and 15% glycerinated PHEM buffer (60 mM PIPES, 25 mM HEPES, 10 mM EGTA, 2 mM MgCl, pH 6.9) containing heavy meromyosin for 10 min and observed under a transmission electron microscope. By electron microscopy with the modified heavy meromyosin decorated reaction, actin filaments were clearly demonstrated around SEF in LSEC.