BACKGROUND: The adherence of microorganisms to host tissue is an important process in the pathogenesis of fungal infection. A protein that shares antigenic and structural homology with the alpha-subunit of leukocyte adhesion glycoprotein CD11b/CD18, also known as iC3b receptor, Mo-1 or Mac-1, has been isolated from the surface of C. albicans. OBJECTIVE: This study was done to observe the changes in the expression of iC3b receptor on C. albicans by glucose or immunosuppressive agents and to elucidate the effect of glucose and anti-iC3b receptor antibodies on adhesion between human dermal microvascular endothelial cells(HDMEC) and C. albicans. METHODS: We utilized immunofluorescence study and immunofluorescent flow cytometry and the binding assay of C. albicans to cultured HDMEC in vitro and blocking assay using monoclonal antibody were performed. RESULTS: Immunofluorescence study and immunofluorescent flow cytometric analysis demonstrated surface expression of iC3b receptor on C. albicans. The expression of surface iC3b receptor on C. albicans in creased in a dose dependent manner with increasing concentrations of glucose, cyclophosphamide and prednisolone. The adherence of C. albicans to HDMEC correlated positively with glucose levels. The adherence of C. albicans to HDMEC decreased significantly by the treatment with anti-iC3b receptor antibodies. CONCLUSION: The results suggest that iC3b receptors should be involved in the adherence of C. albicans to vascular endothelial cells and may be involved in the pathogenesis of hematogenous candidiasis
Antibodies ; Candida albicans* ; Candida* ; Candidiasis ; Complement C3b* ; Cyclophosphamide ; Endothelial Cells ; Endothelium* ; Flow Cytometry ; Fluorescent Antibody Technique ; Glucose ; Glycoproteins ; Humans* ; Immunosuppressive Agents ; Leukocytes ; Prednisolone ; Yeasts*

OBJECTIVETo observe the effects of complement fragment C3f on expression and secretion of collagen I, III and transforming growth factor( TGF)-beta1 in human embryonic lung fibroblast (MRC-5) cells.

METHODSMRC-5 cells were cultured with C3f (the synthetic 17 peptides fragments of complement C3). The extracellular and intracellular expression levels of type I, III collagens and TGF-beta1 in MRC-5 cultures were detected by ELISA and immunohistochemistry, respectively.

RESULTSThe expression levels of type I, III collagen and TGF-beta1 in the supernatant of MRC-5 cultures decreased significantly with the concentrations of C3f as compared with controls (P < 0.05). Also the expression level of TGF-beta1 in MRC-5 cytoplasm reduced significantly as compared with controls (P < 0.05).

CONCLUSIONThe results of present in vitro study showed that the complement fragment C3f could reduce the formation of TGF-beta1 and type I, III collagens in MRC-5 cells, and inhibit the lung tissue fibrosis.

Cell Line ; Collagen Type I ; metabolism ; Collagen Type III ; metabolism ; Complement C3b ; pharmacology ; Fibroblasts ; drug effects ; metabolism ; Humans ; Lung ; cytology ; drug effects ; embryology ; Transforming Growth Factor beta1 ; metabolism

OBJECTIVETo explore the characteristics in CFHR1 concentration and the frequency of CFHR1 gene polymorphisms of patients with type 2 diabetes mellitus (T2DM) based on the high level of complement factor H (CFH) expression among such patients and the similarity between CFHR1 and CFH in terms of sequence and functions.

METHODSFifty T2DM patients and 30 healthy controls were selected. The plasma samples were separated by pI with OFFGEL electrophoresis following solution digestion. Further separation and identification were carried out on a Nano HPLC-Chip-MS/MS system. Differentially expressed proteins were identified by comparison. Enzyme-linked immunosorbent assay (ELISA) was used to validate the result. Genomic DNA of the two groups was extracted. Polymerase chain reaction and sequencing were used to determine the single nucleotide polymorphisms in the 6 exons of the CFHR1 gene.

RESULTSThe CFHR1 level in plasma of T2DM patients were significantly higher than that of the healthy controls (P=2.78× 10). A significant difference in allelic frequencies of rs12406079 of the fifth exon of the CFHR1 gene was found between the two groups (χ=5.692, P=0.017).

CONCLUSIONThe concentration of CFHR1 and frequencies of CFHR1 gene polymorphisms among patients with T2DM differ significantly from healthy subjects. Polymorphisms of the CFHR1 gene are associated with T2DM.

Complement C3b Inactivator Proteins ; genetics ; metabolism ; Diabetes Mellitus, Type 2 ; genetics ; metabolism ; Female ; Genotype ; Humans ; Male ; Middle Aged ; Polymorphism, Single Nucleotide

The macrophage scavenger receptor SR-AI binds to host tissue debris to perform clearance and it binds to bacteria for phagocytosis. In addition, SR-AI modulates macrophage activation through cell signaling. However, investigation of SR-AI signaling on macrophages is complicated due to its promiscuous ligand specificity that overlaps with other macrophage receptors. Therefore, we expressed SR-AI on HEK 293T cells to investigate its ligand binding and signaling. On 293Tcells, SR-AI could respond to E. coli DH5α, leading to NF-κB activation and IL-8 production. However, this requires E. coli DH5α to be sensitized by fresh serum that is treated with heat-inactivation or complement C3 depletion. Anti-C3 antibody inhibits the binding of SR-AI to serum-sensitized DH5α and blocks DH5α stimulation of SR-AI signaling. Further analysis showed that SR-AI can directly bind to purified iC3b but not C3 or C3b. By mutagenesis, The SRCR domain of SR-AI was found to be essential in SR-AI binding to serum-sensitized DH5α. These results revealed a novel property of SR-AI as a complement receptor for iC3b-opsonized bacteria that can elicit cell signaling.
Amino Acid Sequence ; Carrier Proteins ; genetics ; metabolism ; Complement C3b ; metabolism ; Escherichia coli ; immunology ; HEK293 Cells ; Humans ; Macrophage Activation ; Molecular Sequence Data ; Mutagenesis ; NF-kappa B ; genetics ; metabolism ; Phagocytosis ; Serine-Arginine Splicing Factors ; Signal Transduction

To assess the value of immunohistochemical analysis for expressions of C3d, C4d, IgG, IgG4, and CD123 in the diagnosis of autoimmune skin diseases.
 Methods: We investigated the expressions of C3d, C4d, IgG, IgG4, and CD123 in paraffin-embedded, formalin-fixed tissues from 27 lupus erythematosus cases, including 8 discoid lupus erythematosus (DLE) cases, 4 subacute cutaneous lupus erythematosus (SCLE) cases, and 15 systemic lupus erythematosus (SLE) cases. Tissues from 15 dermatomyositis (DM) cases, 15 bullous pemphigoid (BP) cases, and 15 pemphigus cases were examined by immunohistochemical analysis. The differences in expression rates of C3d, C4d, IgG, IgG4, and CD123 between immunohistochemical staining and direct immunofluorescence were compared in the diagnosis of these diseases.
 Results: In the lupus erythematosus group, the positive rates of C3d and C4d deposited along the dermoepidermal junction were 85.2% and 51.9%, respectively. In the dermatomyositis group, the positive rates of C3d and C4d deposited along the dermoepidermal junction were 40% and 0, respectively. The expressions of C3d and C4d in lupus erythematosus tissues were significantly higher than those in DM tissues (P<0.05). The expression of CD123 protein in skin lesions of the lupus group was significantly higher than that in the DM group (P<0.05). In the BP group, the positive rates of C3d and C4d deposited along the dermoepidermal junction were 100% and 86.7%, respectively. In the pemphigus group, the positive rates of C3d and C4d deposited in the intercellular space of keratinocytes were 100% and 60%, respectively. The expressions of IgG and IgG4 in pemphigus tissues were higher than those in BP tissues (P<0.05). And the ratios of IgG4 to IgG in the pemphigus group was significantly higher than that in the BP group (P<0.05).
 Conclusion: The assays of C3d and C4d define an important diagnostic adjunct in evaluation of lupus erythematosus, BP and pemphigus. In some cases, it may even replace the direct immunofluorescence as a diagnostic adjunct. The expression of CD123 possesses certain clinical significance for the differential diagnosis of lupus erythematosus, and IgG4 and IgG expressions have adjunctive diagnostic significance for pemphigus.
Autoimmune Diseases ; Complement C3d ; Complement System Proteins ; Humans ; Immunoglobulin G ; Interleukin-3 Receptor alpha Subunit ; Lupus Erythematosus, Systemic ; Pemphigus

We constructed a recombinant plasmid encoding VP1 gene of O type foot-and-mouth disease virus fused to a molecular adjuvant, goat complement C3d gene. The goat C3d gene was cloned and three copies were tandem-linked with the linker (G4S)2 sequence. VP1 gene of O type foot-and-mouth disease virus was linked to three tandem repeats of C3d through the linker sequence and cloned into pUC19 to obtain the recombinant plasmid pUC19-VP1-C3d3. The VP1-C3d3 fusion gene was then subcloned into the eukaryotic vector pcDNA3.1(+) that had been modified to contain the tissue plasminogen activator (tPA) leader sequence to obtain pcDNA3.1-tPA-VP1-C3d3. HeLa cells were transfected with pcDNA3.1-tPA-VP1-C3d3 by Lipofectamine 2000. Indirect immunofluorescent assay and Western blot assay showed that VP1-C3d3 fusion gene was successfully expressed in HeLa cells. The fusion protein with the expected size 133 kD could be secreted outside the cells. This study laid a good foundation to further research on the novel vaccine against foot-and-mouth disease virus by using goat C3d as a molecular adjuvant to enhance the immunogenicity of VP1.
Animals ; Capsid Proteins ; biosynthesis ; genetics ; Cloning, Molecular ; Complement C3d ; biosynthesis ; genetics ; immunology ; Female ; Foot-and-Mouth Disease Virus ; genetics ; Goats ; HeLa Cells ; Humans ; Immunologic Factors ; biosynthesis ; genetics ; immunology ; Plasmids ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Transfection

OBJECTIVETo construct a replication-defective recombinant adenovirus expressing the fusion gene of neuraminidase (NA) gene in influenza virus A/FM/1/47 and C3d and to evaluate the induced immune efficacy.

METHODSNA-C3d was cloned into shutter vector pAdTrack-CMV, which was cotransformated with adenovirus DNA into E. coli BJ5183. The recombinant adenovirus genomic DNA was generated through homological recombination. The recombinant adenovirus was produced by transfecting 293 cell line with the genomic DNA and the induced immune efficacy in mice were analyzed.

RESULTSThe integration of NA-C3d in the adenovirus genomic DNA and its expression were confirmed by PCR and Western-Blot assays respectively. After intranasal immunization, the serum IgG was induced at a titer of 1: 1000 and 1:100 000 in BALB/c mice at primary and secondary immunization respectively. The vaccinated mice were completely survived when challenged with wide influenza virus.

CONCLUSIONrecombinant adenovirus expressing NA-C3d was successfully constructed and it could induce desired immune efficacy.

Adenoviridae ; genetics ; metabolism ; physiology ; Animals ; Cloning, Molecular ; Complement C3d ; biosynthesis ; genetics ; Genetic Vectors ; genetics ; Immunoglobulin G ; immunology ; Influenzavirus A ; enzymology ; genetics ; Mice ; Mice, Inbred BALB C ; Neuraminidase ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Transfection ; methods ; Virus Replication

We studied the immunogenicity of pseudorabies virus gC DNA vaccination by fusing the murine complement C3d receptor binding domain. First, pseudorabies virus gC gene was linked to four copies of C3d receptor binding domain (M284), and then cloned into the vector pcDNA3.1 to construct the recombinant plasmid sgC-M284. Through the experiment of immunized BALB/c mice, we found that the enzyme linked immunosorbent assay (ELISA) antibody titer for sgC-M284 was 17-fold higher than that for sgC alone, and protective rate of mice was augmented from 25% to 88% after lethal dose PrV (316 LD50) challenge. In addition, the IL-4 levels for sgC-M284 immunization approached that for the pseudorabies virus inactivated vaccine. In conclusion, we demonstrated murine C3d receptor binding domain fusion significantly increased Th2-biased immune response by inducing IL-4 production.
Adjuvants, Immunologic ; physiology ; Animals ; Antibody Formation ; immunology ; Binding Sites ; Cloning, Molecular ; Complement C3d ; genetics ; immunology ; Herpesvirus 1, Suid ; genetics ; immunology ; Interleukin-4 ; immunology ; Mice ; Mice, Inbred BALB C ; Pseudorabies Vaccines ; immunology ; Receptors, Complement 3d ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Swine ; Vaccines, DNA ; immunology ; Viral Envelope Proteins ; pharmacology ; Viral Fusion Proteins ; immunology