Y H Kang's research while affiliated with Walter Reed National Military Medical Center and other places

To produce cell lines that can be used as a continuous source of antigen presenting cells for stimulating T-cell lines and clones and as targets in cytotoxic T-lymphocyte (CTL) assays, we used a retroviral vector with a simian virus (SV40) early promotor to transfer a Plasmodium falciparum circumsporozoite (PfCSP) gene into human EBV transformed B-lymphoblastoid cell lines (B-LCL). We herein report successful, stable transfection and cell surface expression of this gene, as confirmed by PCR, Western blot analysis and immunoelectron microscopy. One of three successfully transfected autologous cell lines expressed PfCSP on the cell surface and was lysed by CD8+ T-cell dependent CTL from a donor volunteer who had been immunized with irradiated P. falciparum sporozoites. Such cell lines should provide excellent tools for characterizing human CD8+ T-cell responses against Plasmodium sp. proteins.

Previous animal studies have demonstrated that following systemic administration phosphorothioate oligodeoxynucleotides (S-ODNs) are primarily excreted by the kidneys and that renal tissue levels of S-ODNs exceed that of other organs. Thus, the kidney may be an ideal target organ for application of antisense S-ODNs in vivo. We examined which cells within the rat kidney have uptake of radiolabeled S-ODNs following intravenous infusion. A 20-base 35S-ODN was infused into 6 adult male Wistar rats. Three animals each were sacrificed 30 min and 4 h after infusion. The kidneys were then removed, fixed, and tissue autoradiography was performed. Similar results were obtained in both groups. The highest level of radioactivity was seen within the proximal tubules. Lower levels of activity were seen within the glomerulus, the parietal epithelial cells of Bowman's space, and distal tubular cells. Very weak activity was also detected within the cells of the loop of Henle and the medullary collecting ducts. These results demonstrated that within the kidney S-ODNs were taken up primarily by proximal tubular cells, with much lower uptake by cells in other segments of the nephron.

In this report, we studied the effects of transforming growth factor (TGF)-beta 1 on lipopolysaccharide (LPS)-induced expression of endothelial cell (EC) adhesion molecules. Confluent human umbilical cord vein EC cultures were stimulated with Escherichia coli LPS and TGF-beta 1, alone or in combination for various times and evaluated for expression of ICAM-1, E-selectin, and VCAM-1 by immunofluorescence and radioimmunoassay. Effects of LPS and/or TGF-beta 1 on cell growth were also studied by 3H-thymidine incorporation. Both LPS and TGF-beta 1 alone stimulated EC expression of the adhesion molecules in a dose-dependent manner. The effects of TGF-beta 1 on LPS induction of the adhesion molecules varied with LPS concentration and treatment time, mode, and duration. Pretreatment with TGF-beta 1 for 24 h greatly augmented LPS induction of ICAM-1 and VCAM-1 expression, but decreased E-selectin expression. TGF-beta 1 also enhanced expression of the adhesion molecules in cells that were pretreated with 1 microgram/mL LPS for 60 min. Concomitant treatment with TGF-beta 1/LPS resulted in significant increases in ICAM-1 but decreases in VCAM-1 expression. TGF-beta 1 effects on LPS induction of the adhesion molecules were more prominent at lower LPS levels (.001, .01 microgram/mL). Both LPS and TGF-beta 1 suppressed thymidine incorporation in a dose-related pattern. These data suggest that TGF-beta 1 has additive and antagonistic effects on LPS induction of the adhesion molecules and that the cell responsiveness to the stimuli in the expression is related to growth condition of the cells. In conclusion, our findings suggest that TGF-beta 1 exhibits pro-inflammatory and anti-inflammatory activities in human endothelial cells and may play an important role in regulating leukocyte adherence and extravasation under LPS-induced inflammatory conditions.

The binding of soluble CD14 (sCD14) to human umbilical vein endothelial cells (HUVEC) was examined in order to understand the role of CD14 in potentiating LPS activity. Both purified sCD14 and [125I]-sCD14 potentiated LPS-stimulated ICAM-1 expression in HUVEC. This potentiation was blocked by anti-CD14 monoclonal antibodies (mAbs) 63D3, UCHM-1 and RM052. Saturation binding assay revealed that [125I]-sCD14 bound to HUVEC with a ligand-acceptor dissociation constant of 290 nM. The binding of [125 I]-sCD14 was inhibited by sCD14 with a sCD14-acceptor dissociation constant of 24 nM. The density of CD14 acceptors was estimated to be 4-8 x 105 sites per cell. The [125I]-sCD14 binding was inhibited by anti-human CD14 mAbs UCHM-1 and RM052 but not 63D3. The bound [125I]-sCD14 could be washed off by acid buffer, pH 3.0, and its localization on the cell membrane was confirmed by light microscopic autoradiography. Based on the previously published description of LPS-sCD14 interactions and our observation that anti-CD14 mAbs inhibiting sCD14-acceptor binding also blocked the potentiation of LPS activity by sCD14, we propose that bridging or crosslinking between the putative LPS-receptor complex with the sCD14-acceptor complex via LPS-sCD14 interactions is the mechanism of CD14-dependent activation of endothelial cells (EC) by LPS. Re-examination of the published data suggests that this mechanism is a universal one for EC, leukocytes and lymphocytes.

In this report, we studied the effects of transforming growth factor (TGF)-+/-1 on lipopolysac-charide (LPS)-induced expression of endothelial cell (EC) adhesion molecules. Confluent human umbilical cord vein EC cultures were stimulated with Escherichia coli LPS and TGF-+/-1, alone or in combination for various times and evaluated for expression of ICAM-1, E-selectin, and VCAM-1 by immunofluorescence and radioimmunoassay. Effects of LPS and/or TGF-+/-1 on cell growth were also studied by 3H-thymidine incorporation. Both LPS and TGF-+/-1 alone stimulated EC expression of the adhesion molecules in a dose-dependent manner. The effects of TGF-+/-1 on LPS induction of the adhesion molecules varied with LPS concentration and treatment time, mode, and duration. Pretreatment with TGF-+/-1 for 24 h greatly augmented LPS induction of ICAM-1 and VCAM-1 expression, but decreased E-selectin expression. TGF-+/-1 also enhanced expression of the adhesion molecules in cells that were pretreated with 1 +/-g/mL LPS for 60 min. Concomitant treatment with TGF-+/-1/LPS resulted in significant increases in ICAM-1 but decreases in VCAM-1 expression. TGF-+/-1 effects on LPS induction of the adhesion molecules were more prominent at lower LPS levels (.001, .01 +/-g/mL). Both LPS and TGF-+/-1 suppressed thymidine incorporation in a dose-related pattern. These data suggest that TGF-+/-1 has additive and antagonistic effects on LPS induction of the adhesion molecules and that the cell responsiveness to the stimuli in the expression is related to growth condition of the cells. In conclusion, our findings suggest that TGF-+/-1 exhibits pro-inflammatory and anti-inflammatory activities in human endothelial cells and may play an important role in regulating leukocyte adherence and extravasation under LPS-induced inflammatory conditions. (C)1996The Shock Society

Endotoxins (lipopolysaccharides; LPS) are known to cause multiple organ failure, including renal dysfunction. The present report elucidates LPS distribution and effect on renal proximal tubules in an attempt to gain a better understanding of the cellular mechanism underlying the pathogenesis of renal dysfunction in endotoxemia and sepsis. Rats were intravenously treated with biotin-linked or regular Escherichia coli (0111:B4) LPS (3 mg/kg) and sacrificed at different times. Kidneys were retrieved and examined for LPS localization, tubular permeability, ultracytochemical alterations, leukocyte sequestration, and ICAM-1 expression. The functional impact of endotoxemia was also assessed by monitoring the changes in urine levels of glucose in timed collections up to 6 h. LPS was localized on the plasma membranes of the apical microvilli, the labyrinth of the lateral intercellular spaces, in various organelles of epithelial cells, and in the endothelial cells of the peritubular capillaries. LPS caused structural damage and calcium accumulation in the mitochondria, leakage of tight junctions, widening of the basolateral intercellular spaces, intracellular and extracellular edema, leukocyte margination and accumulation, vascular expression of ICAM-1, and decrease of plasma membrane and mitochondrial Ca2(+)-ATPase. Physiological study showed that both urine volume and glucose were greatly increased after LPS infusion. The pathological alterations in the proximal tubules may directly contribute to the reduction in the reabsorption ability of the proximal tubules.

Antisense gene suppression has been carried out for human ICAM-1, ELAM-1, and VCAM-1 in cultured human umbilical vein endothelial cells (HUVEC) stimulated by lipopolysaccharide, tumor necrosis factor alpha, or interleukin-1 beta. A panel of antisense phosphorothioate oligodeoxyribonucleotides (PS-ODN), complementary to mRNA or pre-mRNA of these molecules, were tested for their gene suppression activity monitored by radioimmunoassay of the respective cell surface adhesion molecules. Sequences targeted by effective antisense PS-ODNs were located throughout the mRNA and pre-mRNA. "Hot spots" of gene suppression sites for each region were observed. Shift of the PS-ODN hybridizing site upstream or downstream by a few bases resulted in drastic change of gene suppression efficiency. In addition to translation arrest and RNase H activity, a third mechanism was proposed for antisense gene suppression, involving multiple binding sites for PS-ODN and the activities of RNase H and RNases other than RNase H. Suppression of ICAM-1, ELAM-1, or VCAM-1 in HUVEC by their antisense PS-ODNs resulted in the reduction of adhesion of monocytes and U937 to HUVEC. This may suggest cooperativity among the adhesion molecule pairs in endothelial-leukocyte adhesion, since decrease of a single adhesion molecule on EC surface significantly reduced cell-cell adherence.