Biomarkers, Tumor ; CD59 Antigens ; physiology ; Glycosylphosphatidylinositols ; physiology ; Humans ; Membrane Cofactor Protein ; physiology ; Neoplasms ; diagnosis ; therapy

CD55 Antigens ; Complement Activation ; Humans ; Membrane Cofactor Protein ; Pemphigoid, Bullous ; Recombinant Fusion Proteins

OBJECTIVE: To investigate the effect of silencing CD46 and desmoglein 2 (DSG2) in host A549 cells on the entry of human adenovirus type 3 (HAdV-3) and type 7 (HAdV-7) and host cell secretion of inflammatory cytokines. METHODS: RNA interference technique was use to silence the expression of CD46 or DSG2 in human epithelial alveolar A549 cells as the host cells of HAdV-3 or HAdV-7. The binding of the viruses with CD46 and DSG2 were observed with immunofluorescence staining at 0.5 and 1 h after viral infection. The viral load in the host cells was determined with qRT-PCR, and IL-8 secretion level was measured using ELISA. RESULTS: In infected A549 cells, immunofluorescent staining revealed colocalization of HAdV-3 and HAdV-37 with their receptors CD46 and DSG2 at 0.5 h and 2 h after infection, and the copy number of the viruses increased progressively after the infection in a time-dependent manner. In A549 cells with CD46 silencing, the virus titers were significantly lower at 2, 6, 12 and 24 h postinfection in comparison with the cells without gene silencing; the virus titers were also significantly decreased in the cells with DSG2 silencing. The secretion level of IL-8 increased significantly in A549 cells without siRNA transfection following infection with HAdV-3 and HAdV-7 (P < 0.0001), but decreased significantly in cells with CD46 and DSG2 silencing (P < 0.0001). CONCLUSION HAdV-3 and HAdV-7 enter host cells by binding to their receptors CD46 and DSG2, and virus titer and cytokines release increase with infection time. Silencing CD46 and DSG2 can inhibit virus entry and cytokine IL-8 production in host cells.
A549 Cells ; Adenoviruses, Human/metabolism* ; Desmoglein 2/metabolism* ; Humans ; Interleukin-8 ; Membrane Cofactor Protein/genetics* ; RNA, Small Interfering

OBJECTIVETo investigate the inhibitive effects of chimeric oncolytic adenovirus SG235 on leukemia cells in vitro.

METHODSThe ability of SG235 to infect leukemia cells and the expression of CD46 on blasts from the patient with leukemia were detected by flow cytometry (FACS). The cytotoxicity of the virus was evaluated by MTT assay. Apoptosis induced by SG235 was detected with Annexin-V/PI staining and TUNEL assay followed by FACS analysis.

RESULTThe majority of leukemia cells from the patient with acute leukemia was CD46-positive. GFP-positive cells were 45.1%, 35.7%, 54.2%, 37.0%, 30.1%, %67.1, 17.2% and 33.1% in Mutz-1, Kasumi-1, K562, HL60, Molt- 4, RPMI8226, L428, and Jurkat cell lines treated with SG235-EGFP vector at MOI (multiplicity of infection) of 50 for 48 h.SG235 treatment resulted in marked growth inhibition and apoptosis of Kassumi-1 cells, and also significantly inhibited expression of p-Akt.

CONCLUSIONThe chimeric oncolytic adenovirus SG235 can infect leukemia cell effectively and results in the growth inhibition and apoptosis of Kasumi-1 cells in vitro.

Adenoviridae ; genetics ; Apoptosis ; Cell Line, Tumor ; Cell Proliferation ; Genetic Vectors ; Humans ; Leukemia ; genetics ; metabolism ; pathology ; Membrane Cofactor Protein ; metabolism ; Oncolytic Viruses ; Transfection

Measles virus is an enveloped virus with a non-segmented negative-sense RNA genome. Two envelope glycoproteins on the viral surface, namely hemagglutinin (H) and membrane fusion protein (F), are responsible for the virus entry into susceptible host cells. The specific interaction between H and its cellular receptors is a key step in successful virus infection, determining the infectivity and tissue tropism of the measles virus. Thus far, three H receptors have been identified, including the complement regulatory molecule CD46, the signaling lymphocyte activation molecule (SLAM) and the cell adhesion molecule Nectin-4. Here, we reviewed our molecular understanding on the recognition mechanism of these receptors by the viral H protein, aiming to promote future studies on antiviral drug design and measles virus-based oncolytic therapy.
Animals ; Antigens, CD ; metabolism ; Cell Adhesion Molecules ; metabolism ; Hemagglutinins, Viral ; metabolism ; Humans ; Measles virus ; pathogenicity ; physiology ; Membrane Cofactor Protein ; metabolism ; Membrane Fusion ; Membrane Fusion Proteins ; metabolism ; Receptors, Cell Surface ; metabolism ; Receptors, Virus ; metabolism ; Signaling Lymphocytic Activation Molecule Family Member 1

Recombinant expression vector pcDNA3-DAFMCP-DP containing human membrane complement regulatory proteins (hCRPs) decay accelerating factor (DAF) and membrane cofactor protein (MCP) cDNA was constructed by using two independent promoters. After transfected into NIH3T3 cells by calcium phosphate-DNA precipitate method, NIH3T3 pcDNA3-DAFMCP-DP transfectants were obtained by G418 selection. Extraneous genes integration was identified by PCR. The co-expression of human DAF and MCP at both mRNA and protein levels was confirmed by using RT-PCR and Western blot analysis. Human DAF and MCP cDNA were integrated into NIH3T3 pcDNA3-DAFMCP-DP genomic DNA after continuous 30 times passages, indicating that NIH3T3 pcDNA3-DAFMCP-DP were stable cell lines. Human C-mediated cytolysis assays showed that NIH3T3 cells transfected stably with pcDNA3-DAF, pcDNA3-MCP, and pcDNA3-DAFMCP-DP were protected from C-mediated damage and co-expressed human DAF and MCP provided more excellent protection against C-mediated attack, which was compared with either DAF or MCP alone. These results suggest that the dicistronic vector could improve the efficiency of multi-gene delivery and benefit the synergic effect of human membrane complement regulatory proteins DAF and MCP.
3T3 Cells ; Animals ; CD55 Antigens ; biosynthesis ; genetics ; pharmacology ; DNA, Complementary ; genetics ; Drug Synergism ; Graft Rejection ; prevention & control ; Humans ; Membrane Cofactor Protein ; biosynthesis ; genetics ; pharmacology ; Mice ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; pharmacology ; Transfection

OBJECTIVE: To investigate the effect CD36 deficiency on muscle insulin signaling in mice fed a normal-fat diet and explore the possible mechanism. METHODS: Wild-type (WT) mice and systemic CD36 knockout (CD36^-/-) mice with normal feeding for 14 weeks (n=12) were subjected to insulin tolerance test (ITT) after intraperitoneal injection with insulin (1 U/kg). Real-time PCR was used to detect the mRNA expressions of insulin receptor (IR), insulin receptor substrate 1/2 (IRS1/2) and protein tyrosine phosphatase 1B (PTP1B), and Western blotting was performed to detect the protein expressions of AKT, IR, IRS1/2 and PTP1B in the muscle tissues of the mice. Tyrosine phosphorylation of IR and IRS1 and histone acetylation of PTP1B promoter in muscle tissues were detected using co-immunoprecipitation (Co-IP) and chromatin immunoprecipitation (ChIP), respectively. RESULTS: CD36^-/- mice showed significantly lowered insulin sensitivity with obviously decreased area under the insulin tolerance curve in comparison with the WT mice (P < 0.05). CD36^-/- mice also had significantly higher serum insulin concentration and HOMA-IR than WT mice (P < 0.05). Western blotting showed that the p-AKT/AKT ratio in the muscle tissues was significantly decreased in CD36^-/- mice as compared with the WT mice (P < 0.01). No significant differences were found in mRNA and protein levels of IR, IRS1 and IRS2 in the muscle tissues between WT and CD36^-/- mice (P>0.05). In the muscle tissue of CD36^-/- mice, tyrosine phosphorylation levels of IR and IRS1 were significantly decreased (P < 0.05), and the mRNA and protein levels of PTP1B (P < 0.05) and histone acetylation level of PTP1B promoters (P < 0.01) were significantly increased as compared with those in the WT mice. Intraperitoneal injection of claramine, a PTP1B inhibitor, effectively improved the impairment of insulin sensitivity in CD36^-/- mice. CONCLUSION CD36 is essential for maintaining muscle insulin sensitivity under physiological conditions, and CD36 gene deletion in mice causes impaired insulin sensitivity by up-regulating muscle PTP1B expression, which results in detyrosine phosphorylation of IR and IRS1.
Animals ; Gene Deletion ; Histones/genetics* ; Insulin ; Insulin Receptor Substrate Proteins/metabolism* ; Insulin Resistance/genetics* ; Membrane Cofactor Protein/genetics* ; Mice ; Mice, Knockout ; Muscles/metabolism* ; Phosphoric Monoester Hydrolases/metabolism* ; Protein Tyrosine Phosphatase, Non-Receptor Type 1/metabolism* ; Proto-Oncogene Proteins c-akt/metabolism* ; RNA, Messenger/metabolism* ; Receptor, Insulin/metabolism* ; Tyrosine/genetics* ; Up-Regulation

Hyperacute rejection (HAR) is the major obstacle to xenotransplantation. In HAR complement (C') cascade is activated following the binding of xenoreactive antibodies to the donor tissue. Complement receptor type 1 (CR1), the most efficient protein in inhibiting activated C's, was modified with membrane cofactor protein (MCP) to make a more efficient C'-inhibiting hybrid molecule. Modification was done by swapping the four active site short consensus repeats (SCRs) of MCP for the SCRs 8-11 of CR1. The hybrid molecule (CR1/MCP) was expressed on the surface of mouse L cells. When the complement inhibitory activity of the CR1/MCP protein was compared with that of the wild CR1 (wCR 1) protein, CR1/MCP's inhibitory activity was weaker than wCR1's. CR1/MCP protein's L cell protecting activity from complement's attack was more prominent in adherent state than in suspension state. From these results it was suggested that the conformational direction of MCP's inhibitory action on C' is different from that of CR1 and most of the MCP expression seems to be confined to the apical side but not to the basal side of the L cell in adherent state. The wCR1's expression seems to be present on all sides of the L cell. Finally, the inverted direction of SCRS-11 of CR1 or variable length of the serine-threoninrich structure of MCP could be tried to make other CR1/MCP variants with more powerful C' inhibitory activities.
Animals ; Antibodies ; Antigens, CD46 ; Catalytic Domain ; Complement System Proteins* ; Consensus ; Humans ; Mice ; Receptors, Complement ; Tissue Donors ; Transplantation, Heterologous

BACKGROUND: Endogenous complement inhibitors in the brain may protect against the neuroinflammation in Alzheimer's disease. Human neuroblastoma cells were stimulated by Abeta1 - 4 2 to investigate whether the expression of various complement regulator genes is upregulated. METHODS: SK-N-SH cells were incubated overnight with a single dose of 20 microM of Abeta1-42 or 0.5 ng/ml - 5 ng/ml of TNFalpha or both. Actinomycin D (2.5 microM) or cycloheximide (2.5 microM) was also added to the cell suspension. Messenger RNA expression of decay accelerating factor (DAF), membrane cofactor protein (MCP), CD59, complement-receptor 1(CR1), C1 inhibitor (C1-INH), C4-binding protein, factor H, factor I, clusterin and S-protein was measured by RT-PCR. RESULTS: Abeta1-42 and TNFalpha upregulated the expression of C1- INH significantly but increased expression of mRNA for factor H was not statistically significant. The expression of mRNAs for DAF and MCP was at low a level after stimulation. Factor I, CD59 and clusterin were not changed in their mRNA level. The mRNAs for S-protein, C4-binding protein and CR1 were not detected. Actinomycin D suppressed mRNA levels of C1-INH and CD59 significantly. Cycloheximide also inhibited the expression of both C1-INH and CD59. CONCLUSIONS: Early upregulated expression of C1-INH in Abeta1-42 stimulated neuroblastoma cell may contribute to a host defense mechanism against complement-mediated neuronal cell damage.
Alzheimer Disease ; Amyloid beta-Peptides ; Antigens, CD46 ; Antigens, CD55 ; Antigens, CD59 ; Brain ; Clusterin ; Complement Factor H ; Complement System Proteins* ; Cycloheximide ; Dactinomycin ; Fibrinogen ; Genes, Regulator* ; Humans* ; Neuroblastoma* ; Neurons ; RNA, Messenger ; Tumor Necrosis Factor-alpha

BACKGROUND: Endogenous complement inhibitors in the brain may protect against the neuroinflammation in Alzheimer's disease. Human neuroblastoma cells were stimulated by Abeta1 - 4 2 to investigate whether the expression of various complement regulator genes is upregulated. METHODS: SK-N-SH cells were incubated overnight with a single dose of 20 microM of Abeta1-42 or 0.5 ng/ml - 5 ng/ml of TNFalpha or both. Actinomycin D (2.5 microM) or cycloheximide (2.5 microM) was also added to the cell suspension. Messenger RNA expression of decay accelerating factor (DAF), membrane cofactor protein (MCP), CD59, complement-receptor 1(CR1), C1 inhibitor (C1-INH), C4-binding protein, factor H, factor I, clusterin and S-protein was measured by RT-PCR. RESULTS: Abeta1-42 and TNFalpha upregulated the expression of C1- INH significantly but increased expression of mRNA for factor H was not statistically significant. The expression of mRNAs for DAF and MCP was at low a level after stimulation. Factor I, CD59 and clusterin were not changed in their mRNA level. The mRNAs for S-protein, C4-binding protein and CR1 were not detected. Actinomycin D suppressed mRNA levels of C1-INH and CD59 significantly. Cycloheximide also inhibited the expression of both C1-INH and CD59. CONCLUSIONS: Early upregulated expression of C1-INH in Abeta1-42 stimulated neuroblastoma cell may contribute to a host defense mechanism against complement-mediated neuronal cell damage.
Alzheimer Disease ; Amyloid beta-Peptides ; Antigens, CD46 ; Antigens, CD55 ; Antigens, CD59 ; Brain ; Clusterin ; Complement Factor H ; Complement System Proteins* ; Cycloheximide ; Dactinomycin ; Fibrinogen ; Genes, Regulator* ; Humans* ; Neuroblastoma* ; Neurons ; RNA, Messenger ; Tumor Necrosis Factor-alpha