Synthesis of C3, C5, C6, C7, C8, and C9 by Human Fibroblasts

We investigated the ability of human fibroblasts to produce the components of the final common pathway (C3-C9) of complement in vitro by co-culturing an alternative complement activator (agarose beads) with the cells. The test system involved incubation of beads with anti-complement antibodies followed by radioactive-labelled anti-Ig detection antibodies. Subsequently, the beads were examined in a radioimmunoassay. Our results indicate that human fibroblasts produce C3, C5, C6, C7, C8, and C9. A neoepitope selectively expressed on activated C9 was detected, indicating assembly of the terminal complement complex and thus formation of a functional terminal complement pathway by the fibroblasts.     

Electrophoresis of guinea-pig complement components (C3, C5, C6, C7, C8 and C9) on polyacrylamide gel

The electrophoretic mobilities of C3, C5, C6, C7, C8 and C9 of guinea-pig complement were studied using polyacrylamide (PAA) gel as a supporting medium. The haemolytic activity of each complement component was readily revealed as a discrete haemolytic band in blood agar containing intermediate red blood cells and appropriate reagents which was overlayed on the PAA gel plate after electrophoresis. By this method, the relative mobilities of C3, C5, C6, C7, C8 and C9 were determined as 0.19, 0.40, 0.17, 0.27, 0.31 and 0.57 respectively. An additional, distinct, haemolytic C5 band was detected when native guinea-pig serum was run. The relative mobility of the haemolytic activity which was not detected when purified C5 was electrophoresed, was 0.25.     

Human Umbilical Vein Endothelial Cells Synthesize Functional C3, C5, C6, C8 and C9 In Vitro

Human endothelial cells (EC), cultured serum-free, synthesize de novo protein which increasingly bind to agarose beads (an alternative pathway activator), until a plateau phase is reached after 24-48 h. EC synthesize functional C3, C5, C6, C8 and C9, which were detected on co-cultured agarose beads, using relevant polyclonal anti-complement antibodies. Two monoclonal anti-C9 neoepitope antibodies (aE11, poly C9-MA) bound to the co-cultured beads, showing that the terminal complement complex (TCC) (C5b-9) was assembled on the beads. This also suggests that C7 is synthesized. There seems to be a positive correlation between the amount of agarose-bound labelled protein and agarose-bound complement. The results indicate that EC produce and secrete the components for the functional alternative and terminal pathways of complement.     

Isoprenoid quinones of Campylobacter cryaerophila, C. cinaedi, C. fennelliae, C. hyointestinalis, C. pylori, and "C. upsaliensis"

The isoprenoid quinone contents of Campylobacter cryaerophila, C. cinaedi, C. fennelliae, C. hyointestinalis, C. pylori, and "C. upsaliensis" were determined by reverse-phase thin-layer and high-performance liquid chromatography. All six of these recently named Campylobacter species contained menaquinone-6 (MK-6), but only C. hyointestinalis and "C. upsaliensis" contained 2,[5 or 8]-dimethyl-3-farnesyl-farnesyl-1,4-naphthoquinone (*MK-6), a previously described novel menaquinone of the Campylobacter genus. C. cryaerophila, C. cinaedi, C. fennelliae, and C. pylori contained an unidentified quinone (Un-MK-6) with a molecular weight of 580 and a base peak ion of m/e = 225 by mass spectrometry but with chromatographic properties different from those of MK-6. *MK-6 and Un-MK-6 are important chemotaxonomic markers of Campylobacter and Campylobacter-like organisms.     

Chain folding in oligo(oxyethylene)s. A study of the C1, C10, C13, C15, C18 and C21 dialkyl ethers of pentatetracontaethylene glycol

Oligomers of general formulaH(CH₂)_n(OCH₂CH₂)₄₅O(CH₂)_nH, with n in the range 1 to 21, were prepared. Both the oxyethylene and the alkyl blocks are homogeneous. It was shown (by X-ray scattering, IR spectroscopy and differential scanning calorimetry) that annealed crystalline samples have structures in which the oxyethylene blocks are normal to their end-group planes and are either once-folded (n = 18, 21) or unfolded (n = 10, 13, 15). It was also shown that rapidly crystallised samples with n = 10, 13, 15 and 18 have long spacings (by SAXS) intermediate in value between those of the unfolded and once-folded forms.     

Microarray-based identification of thermophilic Campylobacter jejuni,C. coli,C. lari,and C. upsaliensis

DNA microarrays are an excellent potential tool for clinical microbiology, since this technology allows relatively rapid identification and characterization of microbial and viral pathogens. In the present study, an oligonucleotide microarray was developed and used for the analysis of thermophilic Campylobacter spp., the primary food-borne pathogen in the United States. We analyzed four Campylobacter species: Campylobacter jejuni, C. coli, C. lari, and C. upsaliensis. Our assay relies on the PCR amplification of specific regions in five target genes (fur, glyA, cdtABC, ceuB-C, and fliY) as a first step, followed by microarray-based analysis of amplified DNAs. Alleles of two genes, fur and glyA, which are found in all tested thermophilic Campylobacter spp., were used for identification and discrimination among four bacterial species, the ceuB-C gene was used for discrimination between C. jejuni and C. coli, and the fliY and cdt genes were used as additional genetic markers specific either for C. upsaliensis and C. lari or for C. jejuni. The array was developed and validated by using 51 previously characterized Campylobacter isolates. All isolates were unambiguously identified on the basis of hybridization patterns with 72 individual species-specific oligoprobes. Microarray identification of C. jejuni and C. coli was confirmed by PCR amplification of other genes used for identification (hipO and ask). Our results demonstrate that oligonucleotide microarrays are suitable for rapid and accurate simultaneous differentiation among C. jejuni, C. coli, C. lari, and C. upsaliensis.     

Synthesis of Complement Components C5, C6, C7, C8 and C9 in Vitro by Human Monocytes and Assembly of the Terminal Complement Complex

Monocytes cultured under serum-free conditions secreted protein which bound covalently and non-covalently to agarose beads, an activator of the alternative pathway of complement. There was a significantly binding of monoclonal anti-C3c antibodies, polyclonal anti-C5, anti-C6, anti-C7, anti-C8, and anti-C9 antibodies, and of a monoclonal antibody against a neoantigen of polymerized C9 to agarose beads incubated with the monocytes for 24, 48, 72 or 96 h. From these results, we conclude that monocytes produce C5, C6, C7, C8 and C9 that assemble as the terminal complement complex on the surface of the agarose beads. Activation by agarose of the alternative pathway with generation of particle bound C3 and C5 convertases is a prerequisite for the subsequent formation of the terminal complement complex. Whether SC5b-9 or the membrane attack of complement (C5b-9) is formed on the beads will be examined.     

Circulating C3, C4, and C3 Split Products (C3c and C3d) During Normal Pregnancy*

ABSTRACT: The plasma concentrations of the complement components C3 and C4, as well as the split products C3c and C3d, were measured before, during, and after normal pregnancy. Significantly increased values were observed in the C3 and C3d levels in the second and third trimesters of pregnancy. The level of C4 was not significantly affected by pregnancy and C3c could not be detected using electroimmunoassays. These results suggest that the increased C3 split-product levels observed reflected an increased turnover of native C3 rather than activation of the complement cascade.     

Conformationally altered hyaluronan restricts complement classical pathway activation by binding to C1q, C1r, C1s, C2, C5 and C9, and suppresses WOX1 expression in prostate DU145 cells

Linear non-sulfated hyaluronan (HA) does not bind complement proteins yet inhibits their hemolytic function. We have previously induced the complement inhibitory function of HA by heat treatment. However, heated HA readily loses its anti-complementary activity probably due to instantaneous interchain re-association. Here, HA solutions were heated and then freeze-dried. Compared to native HA, heated/freeze-dried HA stably restricted serum complement-mediated hemolysis via the classical pathway, in which serum C1 hemolytic function and C3 activation were blocked. Also, treated HA had a significantly increased binding of component C1q, C1r, C1s, C2, C5, C9, P, D and H. Further, when HA was gel-fractionated by electrophoresis and then freeze-dried, its anti-complementary activity was stably induced. Both native and heated/freeze-dried HA stimulated ERK phosphorylation in prostate DU145 cells. However, treated HA suppressed the expression of tumor suppressors WOX1 and WOX2. Together, HA with an altered conformation stabilizes its inhibition and binding of complement proteins. It may recognize cell surface receptors differently from native HA, thereby differentially regulating the expression of cellular proteins.     

Extended multilocus sequence typing system for Campylobacter coli, C. lari, C. upsaliensis, and C. helveticus

A multilocus sequence typing (MLST) system has been reported previously for Campylobacter jejuni to both differentiate strains and identify clonal lineages. However, sequence variation at the MLST loci prevents its use for closely related Campylobacter species. We describe herein an expanded MLST method to include three clinically relevant Campylobacter species, C. coli, C. lari, and C. upsaliensis, and a fourth Campylobacter species, C. helveticus. The C. coli and C. helveticus methods use the same seven C. jejuni loci (aspA, atpA, glnA, gltA, glyA, pgm, and tkt); however, adk and pgi were substituted for aspA and gltA in C. lari and for gltA and pgm in C. upsaliensis. Multiple C. coli (n = 57), C. lari (n = 20), C. upsaliensis (n = 78), and C. helveticus (n = 9) isolates, representing both clinical and environmental sources, were typed. All four species were genetically diverse: the majority (> 80%) of the isolates had unique sequence types (STs). Using this method, mixed C. lari, C. upsaliensis, and C. helveticus isolates were identified; upon separation, each isolate was shown to contain two strains of the same species with distinct STs. Additionally, the expanded MLST method was able to detect potential lateral transfer events between C. jejuni and either C. coli or C. lari and between C. upsaliensis and C. helveticus. Thus, the expanded MLST method will prove useful in differentiating strains of five Campylobacter species, identifying mixed Campylobacter cultures, and detecting genetic exchange within the genus.     

Human Alveolar Macrophages Synthesize Active Complement Components C6, C7, and C8 in Vitro

We investigated whether serum-free human alveolar macrophage cultures synthesize active C6, C7, and C8. There was a significant binding of polyclonal anti-human C6 antibodies to agarose beads incubated with unstimulated macrophages for 24 or 48 h. Endotoxin stimulation of the macrophages was necessary for significant binding of polyclonal anti-C7 and anti-C8 antibodies to agarose beads co-cultured for 48 or 96 h. Two monoclonal antibodies (poly C9-MA and MCaE11) specific for a neoantigen of polymerized C9 in the terminal complement complex (TCC), bound to beads mainly incubated with endotoxin stimulated macrophages. The MCaE11 was more sensitive than the poly C9-MA in detecting the C9 neoantigen on beads incubated with the macrophages or human serum diluted 1:16. We thus conclude that human alveolar macrophages synthesize active C6, C7, and C9 that together with C5 and C9, assemble as the TCC on co-cultured agarose beads. Activation of the alternative pathway on the agarose with generation of fixed C3 and C5 convertases is a prerequisite for the subsequent generation of the TCC.     

Colony multiplex PCR assay for identification and differentiation of Campylobacter jejuni,C. coli,C. lari,C. upsaliensis,and C. fetus subsp. fetus

A multiplex PCR assay was used to simultaneously detect genes from the five major clinically relevant Campylobacter spp. Those genes selected were hipO and 23S rRNA from Campylobacter jejuni; glyA from each of C. coli, C. lari, and C. upsaliensis; and sapB2 from C. fetus subsp. fetus. The assay was evaluated with 137 clinical and environmental isolates and was found to be rapid and easy to perform and had a high sensitivity and specificity for characterizing isolates, even in mixed cultures.     

Specific PCR Identification and Differentiation of the Thermophilic Campylobacters, Campylobacter jejuni, C. coli, C. lari, and C. upsaliensis

A sensitive PCR assay that detects the thermophilic campylobacters C. jejuni, C. coli, C. lari, and C. upsaliensis is reported. Furthermore, by digestion of the PCR products with two restriction enzymes, species differentiation was demonstrated. Thus, the present method has the potential to be used for both detection and identification of thermophilic Campylobacter species.     

Stress, adrenals, and vitamin C

Large daily doses of vitamin C maintained high levels of corticosteroids in the blood of stressed mice. Of these corticosteroids, glucocorticoid is known to suppress the immune response. It is hypothesized that large doses of vitamin C may reduce the organism's immunity to disease when stress is present.     

Secreted Aspergillus fumigatus Protease Alp1 Degrades Human Complement Proteins C3, C4, and C5

The opportunistic human pathogenic fungus Aspergillus fumigatus is a major cause of fungal infections in immunocompromised patients. Innate immunity plays an important role in the defense against infections. The complement system represents an essential part of the innate immune system. This cascade system is activated on the surface of A. fumigatus conidia and hyphae and enhances phagocytosis of conidia. A. fumigatus conidia but not hyphae bind to their surface host complement regulators factor H, FHL-1, and CFHR1, which control complement activation. Here, we show that A. fumigatus hyphae possess an additional endogenous activity to control complement activation. A. fumigatus culture supernatant efficiently cleaved complement components C3, C4, C5, and C1q as well as immunoglobulin G. Secretome analysis and protease inhibitor studies identified the secreted alkaline protease Alp1, which is present in large amounts in the culture supernatant, as the central molecule responsible for this cleavage. An alp1 deletion strain was generated, and the culture supernatant possessed minimal complement-degrading activity. Moreover, protein extract derived from an Escherichia coli strain overproducing Alp1 cleaved C3b, C4b, and C5. Thus, the protease Alp1 is responsible for the observed cleavage and degrades a broad range of different substrates. In summary, we identified a novel mechanism in A. fumigatus that contributes to evasion from the host complement attack.Aspergillus fumigatus is the most important airborne fungal pathogen. The frequency of invasive mycoses due to this opportunistic fungal pathogen has increased significantly during the last 2 decades (reviewed in references 7 and 42). In healthy individuals, A. fumigatus conidia are inhaled but the establishment of disease is prevented by the host immune system. Inhaled A. fumigatus conidia are immediately confronted with the host complement system and phagocytic cells. The complement system is activated on the conidial and hyphal surface (26), and this activation results in the cleavage of C3. Cleavage products of this central component of the complement cascade act as opsonins on the surfaces of pathogens and enhance phagocytosis by neutrophils, macrophages, and eosinophils (69). Opsonization with complement proteins is important for phagocytosis of A. fumigatus conidia, the key process in the defense against this pathogen (59).Activation of the complement system occurs via three pathways: the alternative pathway (AP), the lectin pathway, and the classical pathway. The AP is activated on microbial surfaces and plays a pivotal role in the clearance of microorganisms (70). Progression of the cascade leads to generation of a C5 convertase, which produces inflammatory C5a anaphylatoxins, and also to the formation of terminal complement complexes (TCC), which can form membrane attack complexes (MAC) and hence pores on target surfaces. C3b surface deposition and MAC formation are important for clearance of bacteria but appear to play a minor role in the defense against fungi. The complement activation system is controlled by fluid-phase and cell surface-bound regulators. We and others showed before (2, 62) that A. fumigatus conidia bind factor H (the central human regulator of the AP), FHL-1, and CFHR1. Factor H acts as a cofactor for the plasma serine protease factor I, which mediates the cleavage of C3b (21, 35, 41). This blocks C3 convertase formation and leads to downregulation or termination of the complement cascade. In contrast to conidia, A. fumigatus hyphae do not bind factor H (2). Instead, they activate complement on their surface (26). Until now a single, nonprotein activity in culture supernatant that inhibits opsonization of the fungal surface by complement proteins has been described (65). The nature of this molecule has not been discovered yet. A. fumigatus inhibition of complement activation is important, since activation of the complement cascade causes toxic and damaging effector functions. Although hyphae are too big to become phagocytosed by macrophages, attraction and activation of neutrophils by C3a and C5a still lead to the destruction of hyphae. Therefore, here we analyzed whether A. fumigatus hyphae use additional strategies to interfere with the human complement system.     

Level of complement activity and components C1, C4, C2, and C3 in complement response to bacterial challenge in malnourished rats.

In experimentally induced malnutrition in rats, there was no significant difference between the measured level of complement activity of the classical pathway (50% hemolytic complement [CH50]) and that of the alternative pathway (ACH50), although the levels of complement components C1, C4, C2, and C3 were depressed significantly. The complement activity showed a temporary elevation with a peak at 2 or 3 days after bacterial challenge with Staphylococcus aureus in rats, and we call this the complement response. After 3 days, CH50 and C3 in the malnourished rats and ACH50, CH50, and C3 in the well-nourished rats showed a significant increase, and C1, C4, and C2 in both groups tended to elevate. On the basis of these observations, the significance of the elevation of C3 in the complement response to bacterial infection showed a strong influence by enhancing the activation of both the classical and the alternative pathways, since C3 is known to be the junction of both complement pathways. In this way, C3 responded to an earlier stage than did the other components and may contribute to maintaining the body defense system against infection.