[Preparation of streptavidin-C3d fusion protein and detection of its function in vitro].

Objective To detect the prokaryotic expression of streptavidin-complement 3d (SA-C3d) fusion protein and verify its function in vitro. Methods The C3d DNA was amplified using C3 cDNA as a template, and the C3d fragment was ligated with the vector plasmid pET-24a-6His-SA-IL15 after the digestion with a one-step cloning method to obtain the SA-C3d prokaryotic expression plasmid. The correctly sequenced plasmid was transformed into expression competent Rosetta to induce protein expression. The target protein was obtained by nickel column affinity chromatography and urea dialyzed refolding. The function of SA was demonstrated by anchoring the biotinylated MB49 cell experiment, and the function of C3d was detected by an experiment that promoted the growth of Raji cells. Results The prokaryotic expression vector of SA-C3d was successfully constructed. The purified target protein was obtained by nickel column purification and dialysis refolding. The protein was specifically bound to biotinylated MB49 cells, which promoted the proliferation of Raji cells in a dose-dependent manner, indicating that the protein SA-C3d had a bifunctional activity. Conclusion The successfully prepared SA-C3d fusion protein can be bound to biotinylated MB49 cells in vitro and promote Raji cell proliferation.

Acute Complement Inhibition Potentiates Neurorehabilitation and Enhances tPA-Mediated Neuroprotection.

Because complement activation in the subacute or chronic phase after stroke was recently shown to stimulate neural plasticity, we investigated how complement activation and complement inhibition in the acute phase after murine stroke interacts with subsequent rehabilitation therapy to modulate neuroinflammation and neural remodeling. We additionally investigated how complement and complement inhibition interacts with tissue plasminogen activator (tPA), the other standard of care therapy for stroke, and a U.S. Food and Drug Administration preclinical requirement for translation of an experimental stroke therapy. CR2fH, an injury site-targeted inhibitor of the alternative complement pathway, significantly reduced infarct volume, hemorrhagic transformation, and mortality and significantly improved long-term motor and cognitive performance when administered 1.5 or 24 h after middle cerebral artery occlusion. CR2fH interrupted a poststroke inflammatory process and significantly reduced inflammatory cytokine release, microglial activation, and astrocytosis. Rehabilitation alone showed mild anti-inflammatory effects, including reduced complement activation, but only improved cognitive recovery. CR2fH combined with rehabilitation significantly potentiated cognitive and motor recovery compared with either intervention alone and was associated with higher growth factor release and enhanced rehabilitation-induced neuroblast migration and axonal remodeling. Similar outcomes were seen in adult, aged, and female mice. Using a microembolic model, CR2fH administered in combination with acute tPA therapy improved overall survival and enhanced the neuroprotective effects of tPA, extending the treatment window for tPA therapy. A human counterpart of CR2fH has been shown to be safe and nonimmunogenic in humans and we have demonstrated robust deposition of C3d, the CR2fH targeting epitope, in ischemic human brains after stroke.SIGNIFICANCE STATEMENT Complement inhibition is a potential therapeutic approach for stroke, but it is not known how complement inhibition would interact with current standards of care. We show that, after murine ischemic stroke, rehabilitation alone induced mild anti-inflammatory effects and improved cognitive, but not motor recovery. However, brain-targeted and specific inhibition of the alternative complement pathway, when combined with rehabilitation, significantly potentiated cognitive and motor recovery compared with either intervention alone via mechanisms involving neuroregeneration and enhanced brain remodeling. Further, inhibiting the alternative pathway of complement significantly enhanced the neuroprotective effects of thrombolytic therapy and markedly expanded the therapeutic window for thrombolytic therapy.

[Expression of NA of influenza virus and C3d fusion gene in replication-defective recombinant adenovirus and its immune efficacy analysis].

OBJECTIVE: To 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. METHODS: NA-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. RESULTS: The 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. CONCLUSION: recombinant adenovirus expressing NA-C3d was successfully constructed and it could induce desired immune efficacy.

Complement factor B expression profile in a spontaneous uveitis model.

Equine recurrent uveitis serves as a spontaneous model for human autoimmune uveitis. Unpredictable relapses and ongoing inflammation in the eyes of diseased horses as well as in humans lead to destruction of the retina and finally result in blindness. However, the molecular mechanisms leading to inflammation and retinal degeneration are not well understood. An initial screening for differentially regulated proteins in sera of uveitic cases compared to healthy controls revealed an increase of the alternative pathway complement component factor B in ERU cases. To determine the activation status of the complement system, sera were subsequently examined for complement split products. We could demonstrate a significant higher concentration of the activation products B/Ba, B/Bb, Bb neoantigen, iC3b and C3d in uveitic condition compared to healthy controls, whereas for C5b-9 no differences were detected. Additionally, we investigated complement activation directly in the retina by immunohistochemistry, since it is the main target organ of this autoimmune disease. Interestingly, infiltrating cells co-expressed activated factor Bb neoantigen, complement split product C3d as well as CD68, a macrophage marker. In this study, we could demonstrate activation of the complement system both systemically as well as in the eye, the target organ of spontaneous recurrent uveitis. Based on these novel findings, we postulate a novel role for macrophages in connection with complement synthesis at the site of inflammation.

[Construction and eukaryotic expression of recombinant plasmid encoding fusion protein of goat complement C3d and foot-and-mouth disease virus VP1].

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.

A simple method for the production of anti-C3d monoclonal antibody.

Production of monoclonal antibodies to C3d usually involves the purification of protein. Our method does not require C3 purification; it relies on attachment of C3b to mouse erythrocytes by activation of alternative pathways and further conversion in C3d. We prepared human complement-coated mouse red cells and sensitized mice of the same strain with our own schedule of immunization and applied the classical methods to obtain a mouse monoclonal antibody. We obtained a clone called BMS-11 which produces a monoclonal antibody of IgM class, to C3d with a title of 1:500000. The monoclonal antibody obtained has shown that it is suitable for use as an antiglobulin reagent.

Cloning of a gene fragment encoding bovine complement component C3d with expression and characterization of derived fusion proteins.

The gene fragment coding for bovine C3d gene (boC3d) was cloned and expressed as a component of fusion proteins destined for use in vaccine studies in cattle, and for in vitro experiments. This fragment of complement protein C3 (C3d) has been shown to enhance B cell responses when complexed with antigen. Three potential vaccine constructs were engineered to contain one, two or three boC3d units linked to a fragment of the leukotoxin of Mannheimia haemolytica A1, an economically important pathogen of cattle that causes a fibrinous pneumonia in calves. A recombinant biotinylated boC3d protein (for use in in vitro studies) was generated by endogenous biotinylation in Escherichia coli by means of the BirA holoenzyme synthetase. All recombinant proteins incorporated polyhistidine tags and were purified by nickel-agarose chromatography, then analyzed by SDS-PAGE and Western immunoblot. The identity of boC3d was confirmed by mass spectrometry, since monoclonal antibodies to boC3d were not available. To date, published research into the adjuvant activities of C3d has been limited to experiments in mice and rabbits, using antigens unrelated to diseases occurring naturally in these species. The boC3d fusion proteins expressed in this study will provide the basis for immunization trials in cattle and studies of receptor binding and cell activation of bovine lymphocytes.

Efficient production of complement (C3d)3 fusion proteins using the baculovirus expression vector system.

Proteins fused to activated complement (C) fragments elicit enhanced immunogenicity. This "natural adjuvant" effect may have important implications when considering novel vaccination approaches. Here we describe both the construction of a novel fusion protein, consisting of a well characterized test antigen fused to multiple copies of the activated complement component (C3d)3, as well as an efficient method for its expression and production in insect cells. Using the inherent biological advantages of the baculovirus expression system, as well as applying specific infection and harvesting modifications, we have optimized the efficiency of protein production. Our modifications allow purification of fusion proteins directly from cell supernatant in a single anion exchange chromatographic step. This alleviates the requirement for the inclusion of protein affinity tags. The integrity of the purified recombinant protein was evaluated by SDS PAGE analysis, reactivity with antibodies, as well as in vivo by administration as an immunogen.

Intraglomerular synthesis of complement C3 and its activation products in IgA nephropathy.

BACKGROUND: Complement activation is thought to be pathologically important in IgA nephropathy (IgAN). Although C3 deposition in the mesangium is found in IgAN, the origin of C3 is not clear. We recently demonstrated intraglomerular C3 synthesis in the human kidney; however, the activation and pathological role of locally synthesized C3 remains unclear. Here we performed nonradioactive in situ hybridization for C3 mRNA and immunohistochemistry for C3 and its activation products, such as C3d and membrane attack complex (MAC), to determine whether locally produced C3 in glomeruli was activated in IgA nephropathy. METHODS: Renal samples from 14 patients with IgAN and 5 with minimal change nephrotic syndrome (MCNS) were examined. Uninvolved portions of surgically removed kidneys with tumors served as normal controls. RESULTS: C3 mRNA was not detected in glomeruli in control tissue and MCNS, but was strongly expressed in resident glomerular cells of IgAN, including mesangial cells, glomerular epithelial cells and the cells of Bowman's capsule. Examination of serial sections disclosed that more than 70% of cells positive for C3 mRNA were also stained for C3 protein, C3d, and MAC. Double staining for in situ hybridization and immunohistochemistry also revealed that those C3 mRNA signals were present in intraglomerular cells positive for C3. The expression of C3 mRNA and MAC in glomeruli correlated significantly with the degree of mesangial matrix expansion. CONCLUSIONS: Our results demonstrated that locally synthesized C3 is activated in the glomeruli of IgAN and that its expression correlated with the severity of mesangial matrix expansion. These findings suggest that activation of C3 may be involved in tissue injury in IgAN through the formation of membrane attack complex.

Complement and the recruitment of mononuclear osteoclasts.

AIM: To investigate the hypothesis that complement mediates the recruitment of mononuclear osteoclast precursors to the exposed mineralised bone surface. METHODS: Synthetic hydroxyapatite was incubated in vitro with fresh human serum, with and without complement activation inhibitors. Assays for complement components and the generation of the C3 breakdown product C3d were done. C3 deposition in human fetal tibia primary spongiosa was localised immunohistochemically and complement receptors CR1, CR2, CR3, and CR4 were localised cellularly. Immunohistochemical and enzyme histochemical characterisation of the mononuclear and multinuclear osteoclasts was made with emphasis on their association with complement C3 deposition. RESULTS: Components of complement bind to synthetic hydroxyapatite crystals and, at lower concentrations, C3d was generated in the fluid phase. C3 was deposited in a focal and linear distribution on newly formed bone trabecular surfaces in the primary spongiosa. In a similar distribution CD61, CD68, and tartrate resistant acid phosphatase positive mononuclear osteoclasts were shown in close apposition to the bone trabecular surface. These mononuclear osteoclasts, unlike multinucleate osteoclasts, expressed the complement receptors CR3 and CR4. CR1 and CR2, however, could not be shown on either mononuclear or multinuclear osteoclasts. CONCLUSION: It is suggested that C3 deposition on mineralised bone surfaces mediates the recruitment of mononuclear osteoclasts to this site. As the mononuclear osteoclasts fuse to form the multinucleate osteoclast, complement receptor expression is lost.

Complement activation and the production of inflammatory mediators during the treatment of severe sepsis in humans.

Sepsis or septic shock is frequently associated with activation of the complement system, coagulation and fibrinolytic changes and the release of several cytokines. In this study we analyzed the relation of complement activation to the inflammatory mediators, hemodynamic and biochemical parameters and severity of illness and outcome in 20 consecutive patients with clinically defined sepsis. Levels of C3a and C3d were elevated in 90% of the patients (median levels 0.19 mg/l and 8.6 mg/l respectively) in comparison to 14% and 42%, respectively of 7 patients with non-septic shock. Levels of C4 were decreased in only 1 of the 20 septic patients. Levels of TNF and IL-6 were elevated in 94% and 100% of the patients, Levels of TNF and IL-6 were elevated in 94% and 100% of the patients, respectively (median levels 122 ng/l and 1300 U/ml) and were clearly interrelated (r = 0.67, p less than 0.01). C3a levels correlated with the APACHE II score (r = 0.57, p less than 0.05) and high C3a levels were associated with fatal outcome (p less than 0.05). C3a was also correlated inversely with mean arterial pressure (r = 0.50, p less than 0.01). Levels of complement C3a and C3d significantly correlated with levels of plasminogen activator inhibitor-1 (PAI) and correlated inversely with AT-III levels. We found no correlation between these complement products and leukocyte counts or lactate levels, nor was there a correlation between C3a or C3d and the cytokines TNF and IL-6. Levels of C3a and C3d did not decrease significantly during the first 24 h of treatment, in contrast to a clear decrease in IL-6 levels in all patients and a decrease in TNF in the surviving patients. TNF levels remained stable or increased in the non-survivors. We conclude that both the complement system and the cytokine system are involved in the pathogenesis of septic shock and may be involved in the development of some of the fatal complications like hypotension and disseminated intravascular coagulation.