Biomedical subjects

Daisuke Fukuta

Publications and source records attributed to Daisuke Fukuta.

6 recordsLinked to original sources

PubMed2006

Effect of tandem forms of DAF(CD55) on complement-mediated xenogeneic cell lysis.

BACKGROUND: It is difficult to produce a transgenic animal with high expression of decay-accelerating factor (CD55: DAF) or other molecules. The purpose of this study was to assess the effect of tandem forms of DAF on a xenogeneic cell membrane against human complement. METHODS: cDNAs of the delta-Short Consensus Repeat (SCR) 1-DAF, the double-DAF, the triple-DAF, and the tetra-DAF with a FLAG-tag were established. Chinese hamster ovary (CHO) cell lines and a pig endothelial cell (PEC) line expressing these molecules were established. The amelioration of complement-mediated lysis by the transfectant molecules on these cells was examined. The CHO cell transfectants were also incubated with normal human serum, and the amount of C3 deposited was determined by FACS analysis. RESULTS: Stable CHO cells and PEC transfectants, in which each molecule was clearly expressed, and Western blots showed that each band corresponded to the expected molecular weight. The extent of amelioration of complement-mediated lysis by these four molecules was then examined. A clear tendency was found, as follows: The higher the tandem number of DAF, the greater was the effect on cytotoxicity. Additional experiments focusing on triple-DAF and tetra-DAF did not indicate any significant difference in complement-mediated lysis. Consistent with the complement-regulatory ability, the inhibitory effect of the deposition of C3 fragments by these molecules was closely related to the degree of amelioration. CONCLUSION: These data indicate that tandem DAF, especially a triple-DAF, is a very effective form for protecting against complement activation.

Animals↗

PubMed2004

Delta-short consensus repeat 4-decay accelerating factor (DAF: CD55) inhibits complement-mediated cytolysis but not NK cell-mediated cytolysis.

NK cells play a critical role in the rejection of xenografts. In this study, we report on an investigation of the effect of complement regulatory protein, a decay accelerating factor (DAF: CD55), in particular, on NK cell-mediated cytolysis. Amelioration of human NK cell-mediated pig endothelial cell (PEC) and pig fibroblast cell lyses by various deletion mutants and point substitutions of DAF was tested, and compared with their complement regulatory function. Although wild-type DAF and the delta-short consensus repeat (SCR) 1-DAF showed clear inhibition of both complement-mediated and NK-mediated PEC lyses, delta-SCR2-DAF and delta-SCR3-DAF failed to suppress either process. However, delta-SCR4-DAF showed a clear complement regulatory effect, but had no effect on NK cells. Conversely, the point substitution of DAF (L147 x F148 to SS and KKK(125-127) to TTT) was half down-regulated in complement inhibitory function, but the inhibition of NK-mediated PEC lysis remained unchanged. Other complement regulatory proteins, such as the cell membrane-bound form factor H, fH-PI, and C1-inactivator, C1-INH-PI, and CD59 were also assessed, but no suppressive effect on NK cell-mediated PEC lysis was found. These data suggest, for DAF to function on NK cells, SCR2-4 is required but no relation to its complement regulatory function exists.

Amino Acid Substitution↗

PubMed2004

Only the light chain is sufficient for the serine protease function of the membrane bound form of factor I on a xeno-surface.

The cell membrane-bound forms of whole factor I (fI-PI), the light chain of the serine protease (SP) domain (SP-PI), and the light chain plus the COOH-terminal 45 amino acid (AA) of the heavy chain (SP+45-PI) were constructed. Chinese hamster ovary (CHO) cells, expressing these molecules were established by transfection of cDNA and confirmed by flow cytometry. Amelioration of complement-mediated cell lysis and complement fragment deposition on the cell surface by the transfectant molecules was tested in each CHO cell by means of a lactate dehydrogenase (LDH) assay and flow cytometry, respectively. A highly expressed fI-PI blocked human complement-mediated cell lysis by approximately 84% of the cells. CHO cell transfectants with SP-PI also showed a clear inhibition in cell lysis by human serum, whereas CHO cell transfectants with SP+45-PI showed no inhibition. In addition, fI-PI and SP-PI, but not SP+45-PI, suppressed C5b-9 deposition on CHO cell surface. These data indicate that the last 45 amino acid of the heavy chain, including a disulfide bridge area, did not participate in the serin protease function of factor I. The results suggest that SP-PI has potential for use in clinical xenotransplantation.

Animals↗

PubMed2003

Effect of hybrid complement regulatory proteins on xenogeneic cells.

To suppress C3 fragment deposition in the classical pathway complement activation on xenogeneic membranes, decay accelerating factor (DAF) was the most effective molecule among the complement regulatory proteins (CRPs) used in the present study. C3 fragment deposition was closely related to subsequent xenogeneic cell lysis. However, other molecules were also very effective in different ways and include phosphatidylinositol (PI)-anchored short consensus repeat (SCR) 2-4 of membrane cofactor protein (MCP-PI), PI-anchored C1 esterase inhibitor (C1-INH-PI), and PI-anchored SCR8-11 of complement receptor type 1 (CR1-PI). On the other hand, regarding a strategy for downregulating C4 fragment deposition, the use of only C1-INH-PI and PI-anchored SCR1-3 of the C4b-binding protein (C4bp-PI) was found to be effective.

Animals↗

PubMed2003

Effect of various forms of the C1 esterase inhibitor (C1-INH) and DAF on complement mediated xenogeneic cell lysis.

The purpose of the present study was to assess the effect of various forms of the surface-bound form of the C1 esterase inhibitor (C1-INH-PI) and decay accelerating factor (DAF) on xenogenic cells. cDNAs of various deletion mutants of the C1-INH-PI, such as delta-1-99 amino acid (AA), delta-108-183AA loop, delta-whole loop, delta-exon5, delta-exon6 + 7, and delta-exon5 + 6 + 7, and that of DAF, the delta-short consensus repeat (SCR) 1-DAF were established. While all deletion mutants of C1-INH-PI except the delta-1-99AA were expressed in the cytoplasm but not on the cell surface, the delta-1-99AA was clearly expressed on the xenogeneic cell surface. Amelioration of complement-mediated xenogeneic cell lysis by delta-1-99AA was next tested, and compared with delta-SCR1 DAF. Both molecules blocked human complement-mediated cell lysis by approximately 57 to 90 and 93 to 98%, respectively, in Chinese hamster ovarian tumor (CHO) cells and pig endothelial cells (PECs). The CHO cell transfectants were incubated with 20% normal human serum, and the amounts of C4 and C3 deposition on the cell surface were analysed by flow cytometry. The DAF transfectant showed a large amount of C4-deposition and much less C3-deposition than the controls (approximately 85% suppression), whereas the delta-1-99AA showed approximately a 40% suppression in both C4- and C3-deposition. Consequently, both the delta-1-99AA C1-INH-PI and delta-SCR1 DAF molecules are quite effective in down-regulating the xenogeneic cell lysis, but accomplished this in different manners.

Animals↗

PubMed2002

Regulation of complement-mediated swine endothelial cell lysis by herpes simplex virus glycoprotein, gC1.

Herpes simplex virus type 1 (HSV-1) encodes several immuno-regulatory proteins that allow it to escape from the human immune system. The regulatory function of a HSV-1 glycoprotein gC (HSV-gC1) molecule on complement-mediated swine endothelial cell (SEC) lysis was investigated. The HSV-gC1 gene was obtained by the PCR method from the HSV-1 genome. The complement-regulatory function of this molecule was analyzed by cytotoxicity assay, using Chinese hamster ovarian tumor (CHO) cell and SEC transfectants and six human serum samples. FACS and Western blot analysis revealed the expression of the HSV-gC1 molecule on the transfectants. The CHO cell transfectants showed significant resistance to cell lysis by the sera that did not contain the anti-HSV-gC1 antibody. The SEC transfectants, however, showed a marked resistance to cell lysis in all cases. The introduction of a viral immune regulator such as HSV-gC1 into the swine cell provides a new approach for successful xenotransplantation.

Animals↗