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

The hemolytic uremic syndrome is a clinical syndrome defined by the presence of thrombocytopenia, microangiopathic hemolytic anemia and acute renal failure. Atypical hemolytic uremic syndrome (aHUS) which is not usually associated with prodromal symptoms, especially diarrhea, has a higher mortality rate and a stronger tendency to progress to chronic renal failure. In approximately 30-50% of patients with aHUS, mutations have been detected in complement factor H, membrane cofactor protein or factor I. Mutations in the complement regulator factor H are the most frequent and have a very poor prognosis, with most patients developing ESRD. We have experienced a 33-year-old man with a family history of renal failure diagnosed as aHUS resulted from factor H mutation, for whom we carried out hemodialysis, plasmapheresis and other conservative management.
Acute Kidney Injury ; Adult ; Anemia, Hemolytic ; Antigens, CD46 ; Complement Factor H ; Complement System Proteins ; Diarrhea ; Fibrinogen ; Hemolytic-Uremic Syndrome ; Humans ; Kidney Failure, Chronic ; Plasmapheresis ; Prodromal Symptoms ; Prognosis ; Renal Dialysis ; Renal Insufficiency ; Thrombocytopenia

BACKGROUND: Photodynamic therapy is a viable option for lung cancer treatment, and many studies have shown that it is capable of inducing cell death in lung cancer cells. However, the precise mechanism of this cell death has not been fully elucidated. To investigate the early changes in cancer cell transcription, we treated A549 cells with the photosensitizer DH-I-180-3 and then we illuminated the cells. METHODS: We investigated the gene expression profiles of the the A549 lung cancer cell line, using a DEG kit, following photodynamic therapy and we evaluated the cell viability by performing flow cytometry. We identified the genes that were significantly changed following photodynamic therapy by performing DNA sequencing. RESULTS: The FACS data showed that the cell death of the lung cancer cells was mainly caused by necrosis. We found nine genes that were significantly changed and we identified eight of these genes. We evaluated the expression of two genes, 3-phosphoglycerate dehydrogenase and ribosomal protein S29. The expressed level of carbonic anhydrase XII, clusterin, MRP3s1 protein, complement 3, membrane cofactor protein and integrin beta 1 were decreased. CONCLUSION: Many of the gene products are membrane-associated proteins. The main mechanism of photodynamic therapy with using the photosensitizing agent DH-I-180-3 appears to be necrosis and this may be associated with the altered production of membrane proteins.
Antigens, CD46 ; Carbonic Anhydrases ; Cell Death ; Cell Line ; Cell Survival ; Clusterin ; Complement System Proteins ; Flow Cytometry ; Gene Expression Profiling ; Gene Expression* ; Lung Neoplasms* ; Lung* ; Membrane Proteins ; Necrosis ; Phosphoglycerate Dehydrogenase ; Photochemotherapy* ; Photosensitizing Agents ; Ribosomal Proteins ; Sequence Analysis, DNA ; Transcriptome*

PURPOSE: Local activation of the complement system plays a role in target organ damage. The aim of our study was to investigate the influence of cyclosporine (CsA)- induced renal injury on the complement system in the kidney. MATERIALS AND METHODS: Mice fed a low salt (0.01%) diet were treated with vehicle (VH, olive oil, 1mL/kg/day) or CsA (30mg/kg/day) for one or four weeks. Induction of chronic CsA nephrotoxicity was evaluated with renal function and histomorphology. Activation of the complement system was assessed through analysis of the expression of C3, C4d, and membrane attack complex (MAC), and the regulatory proteins, CD46 and CD55. CsA treatment induced renal dysfunction and typical morphology (tubulointerstitial inflammation and fibrosis) at four weeks. RESULTS: CsA-induced renal injury was associated with increased the expression of C3, C4d, and MAC (C9 and upregulation of complement regulatory proteins (CD 46 and CD55). Immunohistochemistry revealed that the activated complement components were mainly confined to the injured tubulointerstitium. CONCLUSION: CsA-induced renal injury is associated with activation of the intrarenal complement system.
Animals ; Antigens, CD45/analysis ; Antigens, CD46/analysis ; Antigens, CD55/analysis ; Complement C3/analysis ; Complement C4b/analysis ; Complement Membrane Attack Complex/analysis ; Complement System Proteins/*analysis ; Cyclosporine/*toxicity ; Disease Models, Animal ; Immunity, Innate/drug effects ; Immunoblotting ; Immunohistochemistry ; Immunosuppressive Agents/toxicity ; Kidney/*drug effects/immunology/pathology ; Kidney Diseases/*chemically induced/immunology ; Mice ; Microscopy, Confocal ; Peptide Fragments/analysis

The hemolytic uremic syndrome (HUS) is a rare disease of microangiopathic hemolytic anemia, low platelet count and renal impairment. HUS usually occurs in young children after hemorrhagic colitis by shigatoxin-producing enterohemorrhagic E. coli (D+HUS). HUS is the most common cause of acute renal failure in infants and young children, and is a substantial cause of acute mortality and morbidity; however, renal function recovers in most of them. About 10% of children with HUS do not reveal preceding diarrheal illness, and is referred to as D- HUS or atypical HUS. Atypical HUS comprises a heterogeneous group of thrombomicroangiopathy (TMA) triggered by non-enteric infection, virus, drug, malignancies, transplantation, and other underlying medical condition. Emerging data indicate dysregulation of alternative complement pathway in atypical HUS, and genetic analyses have identified mutations of several regulatory genes; i.e. the fluid phase complement regulator Factor H (CFH), the integral membrane regulator membrane cofactor protein (MCP; CD46) and the serine protease Factor I (IF). The uncontrolled activation of the complement alternative pathway results in the excessive consumption of C3. Plasma exchange or plasma infusion is recommended for treatment of, and has dropped the mortality rate. However, overall prognosis is poor, and many patients succumb to end- stage renal disease. Clinical presentations, response to plasma therapy, and outcome after renal transplantation are influenced by the genotype of the complement regulators. Thrombotic thrombocytopenic purpura (TTP), another type of TMA, occurs mainly in adults as an acquired disease accompanied by fever, neurologic deficits and renal abnormalities. However, less frequent cases of congenital or hereditary TTP associated with ADAMTS-13 (a disintegrin and metalloprotease, with thrombospondin 1-like domains 13) gene mutations have been reported, also. Recent advances in molecular genetics better allow various HUS to be distinguished on the basis of their pathogenesis. The genetic analysis of HUS is important in defining the underlying etiology, predicting the genotype-related outcome and optimizing the management of the patients.
Acute Kidney Injury ; Adult ; Anemia, Hemolytic ; Antigens, CD46 ; Child ; Colitis ; Complement Factor H ; Complement Pathway, Alternative ; Complement System Proteins ; Enterohemorrhagic Escherichia coli ; Fever ; Fibrinogen ; Genes, Regulator ; Genotype ; Hemolytic-Uremic Syndrome* ; Humans ; Infant ; Kidney Transplantation ; Membranes ; Molecular Biology ; Mortality ; Neurologic Manifestations ; Plasma ; Plasma Exchange ; Platelet Count ; Prognosis ; Purpura, Thrombotic Thrombocytopenic ; Rare Diseases ; Serine Proteases ; Thrombospondins